CA2868992C - System and method for multiphasic release of growth factors - Google Patents

Abstract

A system for multiphasic delivery of at least one growth factor at a treatment site comprises a delivery vehicle for releasing at least one growth factor in an initial release profile and a carrier for releasing at least one growth factor in a sustained release profile. The initial release profile releases at least one growth factor over a period of hours to days, wherein the growth factor is released in a large amount initially, with the remainder being released in progressively lower amounts. The sustained release profile releases at least one growth factor over a period of days to weeks, wherein the growth factor is released at a generally constant amount over such period. The system of the invention is particularly suited for applications on bioimplants. The invention also comprises methods and kits for multiphasic delivery of at least one growth factor. The invention also comprises calcium sulphate as a carrier for releasing at least one growth factor in both single and multiphasic systems for delivering at least one growth factor at a treatment site.

Description

CA 2,868,992 Blakes Ref: 75312/00030

2 FIELD OF THE INVENTION

3 [0001] This invention relates to systems and methods for releasing biological substances.

4 In particular, the invention relates to the release of growth factors associated with bioimplants.
More particularly, the invention provides a system and method for producing a multiphasic 6 release profile of at least one growth factor to improve the performance of the bioimplant.

8 [0002] Growth factors (GFs) are peptides and proteins that stimulate the growth and/or 9 differentiation of cells via the interaction of the GFs with specific cell surface receptors. Growth factors play an integral role in the repair and regeneration of tissues and exogenous application 11 of GFs can be used to stimulate the repair of various tissues and organs including bone, 12 cartilage, skin and mucosa and to enhance repair of tissues through the stimulation of 13 angiogenesis at the repair site.
14 [0003] The transforming growth factor beta (TGFI3) superfamily of secreted growth and differentiation factors in mammals has over 30 members. These dimeric proteins are 16 characterized by a conserved seven cystine knot-based structure. They regulate the 17 proliferation, differentiation and migration of many cell types, and have important roles in 18 morphogenesis, organogenesis, tissue maintenance and wound healing. The 19 superfamily of growth factors can be subdivided into several subfamilies including the transforming growth factor beta subfamily, the bone morphogenetic protein (BMP) and growth 21 and differentiation factor (GDF) family (also called the BMP subfamily), and the inhibin and 22 activin subfamily.
23 [0004] The BMP subfamily of the TGFI3 superfamily comprises at least twenty proteins, 24 including BMP-2, BMP-3 (also known as osteogenin), BMP-3b (also known as growth and differentiation factor 10, GDF-10), BMP-4, BMP-5, BMP-6, BMP-7 (also known as osteogenic 26 protein-1, OP-1), BMP-8 (also known as osteogenic protein-2, OP-2), BMP-9, BMP-10, BMP-11 27 (also known as growth and differentiation factor 8, GDF-8, or myostatin), BMP-12 (also known 28 as growth and differentiation factor 7, GDF-7), BMP-13 (also known as growth and 29 differentiation factor 6, GDF-6), BMP-14 (also known as growth and differentiation factor 5, 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 GDF-5), and BMP-15 (for a review, see e.g., Azari et al. Expert Opin Invest Drugs 2 200110:1677-1686).
3 [0005] BMPs have been shown to stimulate matrix synthesis in chondroblasts; stimulate 4 alkaline phosphatase activity and collagen synthesis in osteoblasts, induce the differentiation of early mesenchymal progenitors into osteogenic cells (osteoinduction), regulate chemotaxis of 6 monocytes and mesenchymal cells, and regulate the differentiation of neural cells (for a review, 7 see e.g., Azari et al. Expert Opin Invest Drugs 200110:1677-1686 and Hoffman et al. Appl.
8 Microbiol. Biotech 2001;57:294-308).
9 [0006] One of the many functions of BMP proteins is to induce cartilage, bone, and connective tissue formation in vertebrates. The most osteoinductive members of the BMP
11 subfamily are BMP-2, BMP-4, BMP-6, BMP-7, BMP-8 and BMP-9 (see, e.g., Hoffman et al., 12 Appl. Microbiol Biotech 2001, 57-294-308; Yeh et al., J Cellular Biochem., 2005; 95-173-188;
13 and Boden, Orthopaedic Nursing 2005,24:49-52). This osteoinductive capacity of BMPs has 14 long been considered very promising for a variety of therapeutic and clinical applications, including fracture repair; spine fusion; treatment of skeletal diseases, regeneration of skull, 16 mandibular, and bone defects; and in oral and dental applications such as dentogenesis and 17 cementogenesis during regeneration of periodontal wounds, extraction socket grafting, alveolar 18 ridge augmentation , and sinus augmentation. Currently, recombinant human BMP-2 sold as 19 INFUSE by Medtronic FDA approved for use in spinal fusion surgery, for repair of fracture non-unions and for use in oral surgery, while and recombinant human BMP-7 sold as OP-10 by 21 Stryker is approved as an alternative to autograft in recalcitrant long bone nonunion and for 22 revision posterolateral (intertransverse) lumbar spine fusions, where autograft and bone marrow 23 harvest are not feasible or are not expected to promote fusion.
24 [0007] Other recombinant growth factors that have been used exogenously to enhance bone repair include various TGFr3s (see Clokie & Bell, J. Craniofacial Surg.
2003, 14:268-77), 26 members of the fibroblast growth factor superfamily (FGFs) (see Kawaguchi et al., (2007) J.
27 Orthopaedic Res. 25(4): 480-487), members of the platelet derived growth factor superfamily 28 (PDGFs) (see Hollinger et al., 2008 JBJS 90(s1):48-54), and vascular endothelial growth factor 29 (VEGF) (Street et al., 2002 PNAS 99:9656-61).

23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 [0008] For these growth factors to be effective they must be active and available at a 2 sufficient concentration at the time when critical densities of the appropriate responsive cells are 3 present in the repair site. The short half-life, thermal instability, sensitivity to proteases and/or 4 solubility of the GFs requires their administration in combination with a carrier to achieve this requirement.
6 [0009] A number of carriers have been evaluated for the delivery of GFs. These include 7 fibrous collagen sponges, gelatin hydrogels, fibrin gels, heparin, reverse phase polymers such 8 as the poloxamers, carriers composed of poly-lactic acid (PLA), poly-glycolic acid (PGA) or their 9 co-polymers (PLGA), heparin-conjugated PLGA carriers, and inorganic materials such as calcium phosphates. For example the bioimplant (GEM-21S ) which is used for periodontal 11 regeneration uses beta tricalcium phosphate (l1-TCP) as the carrier for rhPDGF-BB.
12 [0010] However, these carriers are of limited effectiveness, due to loss of growth factor 13 activity when associated with the carrier, inefficient release of the GF
at the implantation site, 14 and/or poor protection from proteolysis and degradation. For example the bioimplant Infuse uses a type I collagen sponge as the carrier for rhBMP-2. The rhBMP-2 is released in a burst 16 from the carrier and the half life of the BMP within the wound site is 1-3 days (Winn et at., 1998, 17 Adv. Drug Del. Rev. 31:303; Friess et. at., 1999, Intl. J.
Pharm.,187:91). By the time the 18 mesenchymal stem cells which regenerate bone have migrated into the wound site only 19 fractions of a percent of the original amount of BMP loaded is present to stimulate these cells to make bone. The current solution to ensure an effective level of BMP remaining at these later 21 times is to significantly increase the amount of BMP that is initially loaded. These increased 22 doses increase the risk of complications including bone formation beyond the implant site, 23 autoimmune responses and potentially cancer. Further this dramatically increases the cost of 24 the implant.
[0011] Therefore, a need exists in the art for materials and methods which release growth 26 factors with a profile which minimizes the amount of growth factor that needs to be loaded to 27 achieve the required therapeutic effect.
28 [0012] One strategy is to encapsulate the GF in a biodegradable polymeric matrix that 29 releases the GF with a sustained release profile over many days. For example BMPs have been combined with poly-lactic acid (PLA) or poly-lactic co-glycolic acid (PLGA) to produce 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 sustained release profiles. However the incorporation of the BMP in the PLA or PLGA can 2 denature the BMP reducing its activity and it is difficult to manipulate the release profile to 3 optimize the effectiveness of the bioimplant. Further the degradation rate of these carriers is 4 typically such that large amounts of GF remain locked away long after healing is complete.
Consequently large amounts of GF need to be loaded into these polymers to ensure sufficient 6 GF is present at the appropriate times.
7 [0013] Another strategy is to chemically immobilize the GF directly onto the surface of 8 carrier. However this may result in partial or complete loss of activity of the GF, and may restrict 9 the GF activity such that only those cells directly in contact with the carrier are able to interact with the GF and respond (see Steinmuller-Nethl, D. et al., Biomaterials, 2006, 27: 4547-56) 11 which could be undesirable as the effect could be limited to the immediate interface with the 12 carrier and not throughout the wound site.
13 [0014] The composition of the carrier can influence delivery of the GF. Calcium sulphate 14 has been considered desirable as a bone substitute and GF carrier because it is osteoconductive, biodegradable, biocompatible and nontoxic (Chen et al., J.
Craniofacial Surg., 16 2010, 21:188-197). However, calcium sulphate is also known to have a rapid degradation rate 17 when added to bone in situ and little osteoinductive capability, which has limited its usefulness 18 in bone implants.
19 [0015] One strategy to manage calcium sulphate degradation in situ has been to control degradation rate by altering crystal structures and adding polymers (e.g., chitosan) to the 21 calcium sulphate implant mixture (Chen et al., supra). Polymer-coating calcium sulphate pellets 22 that have been impregnated with BMP can decrease the speed of resorption of calcium 23 sulphate and increase compressive strength and osteoinduction of the mixture (Chen et al., 24 supra).
[0016] Composites containing hydroxyapatite (HAp), a major mineral component of bone, 26 and calcium sulphate hemihydrate (CSH, plaster of Paris) have been used in orthopedic grafts 27 (e.g., Damien, C et al., J. Biomed. Mat. Res., 1990, 24: 639-654;
Damien, C et al.,Spine, 2002, 28 16S: S50-S58; Parsons, J., et al., Annals N.Y. Acad. Sci. ). When CSH is mixed with sterile 29 saline or water it immediately begins to gel. While in the gel state HAp, growth factors and/or various matrix components can be mixed together with the CSH to form the graft composite, 23530844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 which can be inserted or injected into a bone defect where it sets in situ. In such methods, CSH
2 initially acts as a binder. However, subsequent resorption of calcium sulphate leaves behind a 3 porous matrix with space for bone in-growth, which can be stimulated by the growth factors in 4 the hardened composite. Similarly, compositions for delivering osteogenic proteins including CSH, a porous particulate polymer mixture and an ostogenic protein are known (U.S. patent 6 5,385,887 and U.S. Patent Application Publication No. 2008/0233165). In each of these 7 methods calcium sulphate degradation is required for growth factor release. Therefore, bone 8 regeneration is dependent on the rate of calcium sulphate degradation.
9 [0017] Bone grafts containing particulate bone and a biocompatible solid component comprising CSH and a calcium phosphate product are known, but do not involve using the CSH
11 or calcium phosphate as a growth factor carrier (U.S. Patent Application Publication No.
12 2011/0208305).
13 [0018] In nature during wound healing multiple GFs are present within the wound site and 14 surrounding tissue at varying concentrations at different times. For example, immediately following bone fracture, platelets at the injury site will initially release large amounts of PDGF, 16 with a sharp decline in protein levels within the fracture site over the following days (see Tyndall 17 et al., Clinical Orthopedics and Related Research, 2003, 408: 319-330).
Conversely BMP-2 is 18 expressed at all stages of the fracture healing process (see Rasubala et al. British Journal of 19 Oral and Maxillofacial Surgery, 2003, 41:173-178), although the amount of BMP-2 varies over time (see Meyer et al. J Bone Jt. Surg 2003, 85-A: 1243-1254). The concentration of these 21 growth factors is estimated to be orders of magnitude lower than those used during therapeutic 22 application of exogenous GF due to matching of the concentration to the cellular requirements 23 and synergistic effects of the multiple growth factors. Producing a system that allows the 24 delivery of growth factors with multiphasic release profiles and the release of multiple growth factors with different release profiles would permit the use of bioimplants with GF release 26 profiles that more closely mimic GF release during the natural healing process than current 27 bioimplants that release a single growth factor in a burst or with sustained release.
28 [0019] This background information is provided for the purpose of making known 29 information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding 31 information constitutes prior art against the present invention.

5 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 2 [0020] The present invention provides, in one aspect, a system, method and kit for the 3 multiphasic release of at least one growth factor at, for example a treatment site. For this 4 purpose, the system of the invention may be provided as a bioimplant or the like. In one aspect, the method of the invention delivers at least one growth factor in an initial release followed by

6 the delivery of at least one growth factor in a "sustained release profile''. The invention utilizes a

7 delivery system for the initial release and a carrier for the sustained release.

8 [0021] In one aspect, the same growth factor is released in the initial and sustained release

9 profiles. In another aspect, different growth factors are released, with a first growth factor released in an initial profile and a second growth factor released in a sustained release profile.
11 As will be known to persons skilled in the art, the release of two different growth factors in such 12 differing manners is believed to more closely mimic the natural growth factor release system at 13 a treatment site.
14 [0022] In accordance with one aspect of the invention, there is provided a carrier that provides a sustained release of at least one growth factor, combined with a delivery vehicle that 16 provides an initial release of at least one growth factor. The combination of the carrier and the 17 delivery vehicle results in a multiphasic release profile of the growth factor(s). In preferred 18 embodiments, the amount of carrier and delivery vehicle are varied to control the release of at 19 least one GF, wherein the amount of the delivery vehicle and the carrier are provided in a ratio of about 0.5 to 4.0:1(v:v). In preferred embodiments, the amount of delivery vehicle used is 21 between 0.5 and 10.0 ml. In particularly preferred embodiments, 0.75-2.5 ml of delivery vehicle 22 are used with 1cm3 of carrier. In particularly preferred embodiments, 1.0 ml of delivery vehicle 23 and 0.5cm3 of carrier are used.
24 [0023] In preferred embodiments the growth factor ("GF") is a member of the transforming growth factor beta (TGFI3) superfamily. In particularly preferred embodiments the growth factor 26 is a bone morphogenetic protein (BMP).
27 [0024] In one aspect of the present invention, the carrier ("CAR") is formed of calcium 28 phosphate particles with a size less than 80 microns and preferably less than 45 microns 29 dispersed within a polymer matrix which results in a larger structure.
In one aspect, the structure is further coated with a hydroxyapatite layer.

23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 [0025] In one embodiment the at least one GF is/are applied as a liquid to the calcium 2 particles and are then lyophilized onto the particles before combining with the polymer matrix. In 3 some embodiments, 100% of the lyophilized GF is associated with the particles. In other 4 embodiments less than 100% of the lyophilized GF is associated with the particles and the remainder is not associated with the particles. Such composition comprising GF-associated 6 particles and lyophilized GF that is not associated with particles can be combined with a delivery 7 vehicle such that the unassociated particles are distributed in the delivery vehicle, where they 8 can subsequently be released.
9 [0026] In another aspect of the present invention the carrier is formed by mixing one or more calcium phosphate powders with a liquid solution containing at least one growth factor to 11 produce a calcium phosphate cement. In one aspect, the cement is then ground into particles 12 with a diameter of at least 100 microns and preferably between 0.3 and 3mm in diameter.
13 [0027] In another aspect of the present invention the carrier comprises particles of one or 14 more calcium salts all with a diameter of at least 100 microns and preferably between 0.3 and 3mm. A growth factor is then lyophilized onto the surface of the carrier particles.
16 [0028] In preferred embodiments the delivery vehicle is a reverse phase polymer. In 17 particularly preferred embodiments the reverse phase polymer is a poloxamer, more particularly 18 poloxanner 407 (also called PluronicTM F127) at a concentration of at least 12% and preferably 19 between 20 and 40%. In some particularly preferred embodiments, the amounts of P407 and carrier are varied to influence the amount of GF released from the carrier and optionally from 21 the delivery vehicle.
22 [0029] As indicated above, in one aspect, the carrier and the delivery vehicle release the 23 same growth factor while in another aspect, the carrier and delivery vehicle release different 24 growth factors. In yet another aspect of the invention, the carrier and delivery vehicle are each adapted to release combinations of two or more growth factors, with the combination released 26 by each being the same or different.
27 [0030] Thus, in one aspect, the invention provides a system for multiphasic release of 28 growth factors at a treatment site, the system comprising:
29 - a delivery vehicle comprising at least one first growth factor; and - a carrier comprising at least one second growth factor;

23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 -wherein:
2 - the delivery vehicle is adapted to release the at least one first growth factor in an 3 initial release profile over a first time period;
4 - the carrier is adapted to release the at least one second growth factor in a sustained release profile over a second time period.
6 [0031] In another aspect, the invention provides a method of multiphasic release of growth 7 factors, the method comprising:
8 - delivering at least one first growth factor with an initial release profile;
9 - delivering at least one second growth factor in a sustained release profile.
[0032] In a further aspect, the invention provides a kit for multiphasic delivery of growth 11 factors, the kit comprising:
12 - a delivery vehicle component;
13 - at least one first growth factor associated with the delivery vehicle;
14 - a carrier component; and - at least one second growth factor associated with the carrier.
16 [0033] In still a further aspect, the invention provides a kit for multiphasic delivery of growth 17 factors, the kit comprising:
18 - a delivery vehicle component;
19 - a carrier component;
- at least one first growth factor that is not associated with the delivery vehicle or the 21 carrier; and 22 - at least one second growth factor associated with the carrier.
23 [0034] In one embodiment, the kit comprises at least two containers, wherein the first 24 container comprises the delivery vehicle and the second container comprises the carrier associated with the at least one second growth factor and the at least one first growth factor. In 26 preferred embodiments, the at least one first growth factor mixes with the delivery vehicle when 27 the delivery vehicle is added to the carrier.
28 [0035] The present invention also provides, in one aspect, a system, method and kit for the 29 release of at least one growth factor, for example at a treatment site, wherein calcium sulphate 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 "carrier" particles house the at least one growth factor on their surface. For this purpose, the 2 system of the invention may be provided as a bioimplant or the like.
3 [0036] In one aspect, the method of the invention delivers the at least one growth factor in a 4 "sustained release profile'.
[0037] In one aspect of the present invention, the carrier comprises a mixture of calcium 6 sulphate dihydrate and calcium phosphate particles. In preferred embodiments, the ratio of 7 calcium sulphate to calcium phosphate particles is about 1:1 or 2:1.
8 [0038] In some aspects of the present invention, the at least one growth factor is released 9 in a single phase from the calcium carrier. In this aspect, GF is not released by the delivery vehicle. In other aspects, the at least one growth factor undergoes multiphasic release from the 11 calcium carrier and the delivery vehicle. In preferred embodiments, the amount of carrier and 12 delivery vehicle are varied to control the release of at least one GF, wherein the amount of the 13 delivery vehicle and the carrier are provided in a ratio of about 0.5-4:1(v:v). In preferred 14 embodiments, the amount of delivery vehicle used is between 0.5 and 10.0 mi. In particularly preferred embodiments, 0.5-2.5 ml of delivery vehicle is used per cm3 of carrier. In particularly 16 preferred embodiments, 1.0 ml of delivery vehicle and 0.5cm3 of carrier are used.
17 [0039] In one embodiment the at least one GF is/are applied as a liquid to the calcium 18 particles and are then lyophilized onto the particles before combining with the polymer matrix.
19 [0040] In one embodiment the GF is lyophilized such that some of the GF is associated with the carrier and some of the GF is separate from the carrier. When the delivery vehicle is added 21 to carrier the separate GF becomes associated with delivery vehicle.
22 [0041] In preferred embodiments, distribution of the at least one GF onto the calcium 23 particles is altered by varying the volume of the solution containing the GF relative to the protein 24 to be lyophilized onto the particles. The amount of bound GF on calcium particles can be made higher by decreasing the volume of solution used to deliver the GF. In preferred embodiments, 26 lyophilization of GF onto carrier particles is carried out in a 1:1:0.5 ratio, wherein 1 unit of GF is 27 mixed with 1 unit of solution and lyophilized onto 0.5 units of carrier.
In particularly preferred 28 embodiments, about 1.0 mg of GF is added to about 1.0 ml of solution for lyophilization onto 29 about 0.5 cm3 of calcium particles.

23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 [0042] In a further aspect the invention provides a kit for delivery of growth factors, the kit 2 comprising:
3 - a delivery vehicle component;
4 - a carrier component comprising a plurality of calcium sulphate particles;
- at least one second growth factor associated with the carrier and optionally, 6 - at least one first growth factor not associated with the carrier which will become 7 associated with the delivery vehicle when the delivery vehicle is mixed with the at least one first 8 growth factor 9 [0043] In preferred embodiments, the carrier component of the kit further comprises calcium phosphate particles. In a particularly preferred embodiment, the ratio of calcium sulphate to 11 calcium phosphate particles is about 1:1 or 2:1.
12 [0044] In a further aspect the invention provides a kit for multiphasic delivery of growth 13 factors, the kit comprising:
14 - a delivery vehicle component;
- at least one first growth factor associated with the delivery vehicle;
16 - a carrier component comprising a plurality of calcium sulphate particles; and 17 - at least one second growth factor associated with the carrier.
18 [0045] In preferred embodiments, the carrier component of the kit further comprises calcium 19 phosphate particles. In a particularly preferred embodiment, the ratio of calcium sulphate to calcium phosphate particles is about 1:1 or 2:1.

23 [0046] The invention will now be described with reference to the appended figures, which 24 are briefly described below.
[0047] Figure 1 illustrates a sustained release profile exhibited by the carrier of the 26 invention.
27 [0048] Figure 2 illustrates the initial release profile exhibited by the delivery vehicle of the 28 invention.
23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 [0049] Figure 3 illustrates differing release profiles based on an amount of growth factor in 2 the delivery vehicle and carrier. A multiphasic release profile is observed when growth factors 3 are incorporated into both the delivery vehicle and carrier (50-50).
4 [0050] Figure 4 illustrates the in vivo activity of the bioimplants where a growth factor is released as shown in Figure 3 according to the method of the invention.
6 [0051] Figure 5 illustrates the formation of new bone (Bone) onto calcium phosphate 7 particles (CaP) when a bioimplant produced according to the method of the invention was 8 implanted into a mouse.
9 [0052] Figure 6 illustrates the histological appearance of the new bone (Bone) formed on a carrier (Carrier) when bioimplant produced according to the method of the invention was 11 implanted into a mouse.
12 [0053] Figure 7 illustrates a short sustained growth factor release profile produced by a 13 carrier produced according to the method of the invention.
14 [0054] Figure 8 illustrates how a sustained release profile can be altered by changing the properties of the carrier produced according to the method of the invention.
16 [0055] Figures 9A-C illustrate lyophilized carriers, wherein the volume of solution lyophilized 17 was varied but the total protein lyophilized was fixed. Treatment groups 1 (Fig. 9A), 3 (Fig. 9B) 18 and 5 (Fig. 90) are depicted.
19 [0056] Figures 10A-B illustrate histological appearance of new bone formed around calcium phosphate (Fig. 10A) and calcium sulphate (Fig. 10B) carrier components used in a bioimplant 21 produced according to the method of the invention.

23 [0057] Growth factors (GF) play an integral role in the repair and regeneration of tissues 24 and exogenous GFs can be used to stimulate the repair of various tissues and organs. For exogenous growth factors to be effective in stimulating repair they must be retained at the site 26 requiring repair, and be protected from inactivation, sequestration or degradation. To achieve 27 this carriers are used. However the release of growth factors from known carriers is not ideal 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 and cannot be easily modified. The current invention is based on: i) the discovery that the 2 multiphasic release of growth factors from a bioimplant increases the efficacy of the implant;
3 and ii) the discovery that the use of calcium sulphate as a growth factor carrier can improve the 4 potency of GF containing bioimplants.
[0058] The present inventors have developed methods and materials for enhancing the 6 efficacy of, for example, bioimplants by improving the release kinetics or release profile of 7 growth factors at sites of implantation, while maintaining the activity of the growth factors. In 8 one aspect, the present invention provides a growth factor delivery system and method 9 comprising a carrier containing at least one growth factor, combined with a delivery vehicle also containing at least one growth factor. The at least one growth factor released by the carrier and 11 delivery vehicle may be the same or different.
12 [0059] In another aspect, the present invention provides a growth factor delivery system 13 and method that has enhanced efficacy due to using a carrier comprising a plurality of calcium 14 sulphate dihydrate particles comprising at least one GF on their surface, combined with a delivery vehicle that may optionally contain at least one GF. In contrast, previous attempts to 16 use calcium sulphate as a GF carrier involved incorporating or impregnating calcium sulphate 17 particles with the growth factor rather than coating the surface of the calcium particles with a 18 GF. In the present invention, calcium sulphate degradation is not required for GF release.
19 [0060] In preferred embodiments, the amount of carrier and delivery vehicle are varied to control the release of at least one GF, wherein the amount of the delivery vehicle and the carrier 21 are provided in a ratio of about 0.5-4.0:1 (v:v). In preferred embodiments, the amount of 22 delivery vehicle used is between 0.5 and 10.0 ml. In particularly preferred embodiments, 0.5-23 2.5 ml of delivery vehicle is used per cm' of carrier. In particularly preferred embodiments, 1.0 24 ml of delivery vehicle and 0.5 crn3 of carrier are used.
[0061] In one embodiment the at least one GF is/are applied as a liquid to small (<80 26 micron)calcium particles and are then lyophilized onto the particles before combining with a 27 polymer matrix to produce the carrier structure.
28 [0062] In another embodiment the GF is applied as a liquid to large (>100micron) particles 29 and then lyophilized with the particles resulting in a distribution of particle-associated and particle-free GF.

23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 [0063] In preferred embodiments, distribution of the at least one GF between being 2 associated with the particles and being separate or "free" from the particles is altered by varying 3 the volume of the solution containing the GF relative to the amount of particles with which it is 4 incubated prior to lyophilization. The amount of bound GF on particles can be made higher by decreasing the volume of solution used to deliver the GF. In preferred embodiments, 6 lyophilization of GF onto carrier particles is carried out in a 1:1:0.5 ratio, wherein 1 unit of GF is 7 mixed with 1 unit of solution and lyophilized onto 4 units of carrier. In particularly preferred 8 embodiments, about 1.0 mg of of GF is added to about 1.0 ml of solution for lyophilization onto 9 about 0.5cm3 of calcium particles.
[0064] The system and method of the invention can be used for a variety of therapeutic and 11 clinical applications, including: fracture repair; bone grafts; spine fusion; and regeneration of 12 skull, mandibular, and bone defects. For such applications, the system of the invention is 13 preferably provided on, or in the form of a bioimplant.
14 [0065] Definitions [0066] Unless defined otherwise below, all technical and scientific terms used herein have 16 the same meaning as commonly understood by one of ordinary skill in the art to which this 17 invention belongs.
18 [0067] As used herein the term "bioimplant" refers to a material which is suitable for 19 implantation and contains an exogenous growth or biologically active factor. As discussed further herein, the system of the present invention is preferably used by applying same to a 21 bioimplant. The bioimplant is then provided within a body of a subject wherein the system 22 releases at least one growth factor in a multiphasic release profile.
23 [0068] As used herein the term "growth factor" refers to peptides and proteins that stimulate 24 the growth and/or differentiation of cells via the interaction of the GFs with specific cell surface receptors. Examples of growth factors include the bone morphogenetic proteins (BMPs), 26 transforming growth factor beta (TGF3), the insulin-like growth factors (IGF), the fibroblast 27 growth factors (FGFs), platelet derived growth factor (PDGF) and vascular endothelial growth 28 factor. In preferred embodiments the growth factors are BMPs.

23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 [0069] By "recombinant" is meant a protein produced by a transiently transfected, stably 2 transfected, or transgenic host cell or animal as directed by an expression construct containing 3 the cDNA for that protein. The term "recombinant" also encompasses pharmaceutically 4 acceptable salts of such a polypeptide [0070] As used herein, the term "polypeptide" or "protein" refers to a polymer of amino acid 6 monomers that are alpha amino acids joined together through amide bonds.
Polypeptides are 7 therefore at least two amino acid residues in length, and are usually longer. Generally, the term 8 "peptide" refers to a polypeptide that is only a few amino acid residues in length. A polypeptide, 9 -- in contrast with a peptide, may comprise any number of amino acid residues. Hence, the term -- polypeptide included peptides as well as longer sequences of amino acids.
11 [0071] As used herein, the terms "bone morphogenetic protein" or "bone morphogenic 12 protein" or "BMP" are used interchangeably and refer to any member of the bone 13 morphogenetic protein (BMP) subfamily of the transforming growth factor beta (TGFp) 14 superfannily of growth and differentiation factors, including BMP-2, BMP-3 (also known as -- osteogenin), BMP-3b (also known as growth and differentiation factor 10, GDF-10), BMP-4, 16 BMP-5, BMP-6, BMP-7 (also known as osteogenic protein-1, OP-1), BMP-8 (also known as 17 osteogenic protein-2, OP-2), BMP-9, BMP-10, BMP-11 (also known as growth and 18 differentiation factor 8, GDF-8, or myostatin), BMP-12 (also known as growth and differentiation 19 factor 7, GDF-7), BMP-13 (also known as growth and differentiation factor 6, GDF-6), BMP-14 -- (also known as growth and differentiation factor 5, GDF-5), and BMP-15.
21 [0072] The terms "bone morphogenetic protein" and "BMP" also encompass allelic variants 22 of BMPs, function conservative variants of BMPs, and mutant BMPs that retain BMP activity.
23 The BMP activity of such variants and mutants may be confirmed by any of the methods well 24 -- known in the art (see the section Assays to measure BMP activity, below) or as described in Example 1 26 [0073] In preferred embodiments, the BMP is BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, 27 BMP-8 or BMP-9. In particularly preferred embodiments the BMP is BMP-2, BMP-4 or BMP-7.
28 [0074] In preferred embodiments the BMP is a mammalian BMP (e.g., mammalian BMP-2 29 or mammalian BMP-7). In particularly preferred embodiments, the BMP is a human BMP
(hBMP) (e.g. hBMP-2 or hBMP-7).

23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 [0075] As used herein the term "scaffold" refers to a material whose purpose is to provide a 2 structure which supports cell adhesion, migration and ingrowth into a tissue repair site.
3 [0076] As used herein the term "carrier" refers to a material comprising single or multiple 4 components and is adapted to release at least one growth factor at a treatment site in a "sustained release" profile over a period of time. In one aspect, the period of time taken by the 6 carrier to release the at least one growth factor is between several days and several weeks.
7 Preferably, the carrier is adapted to release the at least one growth factor over a period of 8 weeks.
9 [0077] In preferred embodiments the carrier also acts as a scaffold or matrix. As discussed above, in one aspect of the invention, the carrier is formed of calcium phosphate particles 11 dispersed within a macroporous polymer scaffold or matrix. In one aspect, the scaffold or matrix 12 is further coated with a hydroxyapatite layer. In another aspect of the invention, the carrier is 13 formed of calcium sulphate particles. In yet another aspect of the invention, the carrier is a 14 mixture of calcium sulphate and calcium phosphate particles. In one embodiment the at least one growth factor is applied as a liquid to the calcium particles and then lyophilized onto the 16 particles before combining the particles with the polymer matrix. In preferred embodiments, the 17 carrier is a solid.
18 [0078] As used herein the term "delivery vehicle" refers to a material which serves to 19 transport the carrier. In one aspect of the invention, the delivery vehicle comprises or becomes associated with at least one growth factor and is adapted to release the at least one growth 21 factor at a treatment site in an initial release profile over a time period. In other aspects, the 22 delivery vehicle does not initially comprise a growth factor. Rather, it is subsequently combined 23 with a GF that is not associated with the carrier prior to use, thereby producing the initial phase 24 of GF release. In one aspect, the period of time taken by the delivery vehicle to release the at least one growth factor is between several hours and several days. In a preferred embodiment 26 of the invention, the delivery vehicle releases the majority of the at least one growth factor in an 27 "initial release" or "initial release profile" that lasts a period of hours. Preferably, the delivery 28 vehicle is adapted to release at least 80% of the growth factor(s) contained therein (or 29 associated therewith) within a period of 72 hours. In preferred embodiments, the delivery vehicle is a liquid or a gel.
23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 [0079] In one aspect, the delivery vehicle of the present invention may be used to ease 2 handling of the carrier particles, wherein the combination of the carrier and delivery vehicle 3 results in the formation of a gel or putty.
4 [0080] In one aspect, the material forming the delivery vehicle is in the form of a gel. In preferred embodiments the delivery vehicle is a reverse phase polymer. As used herein the 6 term "reverse phase" refers to the property whereby the polymer undergoes a reversible 7 temperature dependent transition from a liquid to a gel. In one aspect the transition temperature 8 is between 15 C and 37 C. Preferably the transition temperature is between 15 C and 25 C.
9 As would be known to persons skilled in the art, "normal phase" materials increase their viscosity with a decline in temperature. In contrast, reverse phase materials show a decline in 11 viscosity as the temperature drops below their transition temperature.
12 [0081] In particularly preferred embodiments the reverse phase polymer is a poloxamer, 13 more particularly Pluronic TM F127 (also known as poloxanner 407 or P407).
14 [0082] In particularly preferred embodiments the P407 polymer solution is between 20 and 40%
16 [0083] In preferred embodiments, the amount of carrier and delivery vehicles used in a 17 bioimplant are altered to influence the amount of GE released from the bioimplant.
18 [0084] As used herein the term "sustained release" or "sustained release profile" refers to 19 the release of at least one growth factor, by the carrier, over a period of several days or weeks with the amount released over an initial period being similar to or less than the amount released 21 over the same period after several days or weeks of implantation.
Preferably, a sustained 22 release profile lasts at least one week. As will be understood by persons skilled in the art, 23 typically, the amount of growth factor released in a sustained release profile over the first three 24 days will be less than the amount released over the following seven days.
[0085] As used herein the term "initial release" or "initial release profile" refers to the initial 26 release, by the delivery vehicle, of a large amount of at least one growth factor followed by 27 progressively smaller amounts released over a period of hours or days.
In one aspect, an initial 28 release profile results in the delivery of at least 80% of the loaded growth factor(s) within a 29 period of roughly 72 hours. An initial release profile is illustrated in Figure 2.

23580844.2 CA 2,868,992 Slakes Ref: 75312/00030 1 [0086] As used herein the term "multiphasic release" refers to an initial release of the at 2 least one growth factor over an initial period of time, followed by "sustained" release of the at 3 least one growth factor over a second period of time. Preferably, the initial period of time is 4 roughly several hours and the second period of time is roughly several days to weeks. Such a release profile may also be referred to as "biphasic release" since it occurs in two stages. In 6 preferred embodiments, the initial release is provided by the delivery system of the invention 7 and the "sustained" release is provided by the carrier of the invention.
8 [0087] As used herein, the term "potency" refers to a measure of drug activity expressed in 9 terms of the amount required to produce an effect of given intensity [0088] In one aspect of the invention, the delivery vehicle component comprises at least 11 10% and not more than 50% of the total amount of growth factor(s) delivered by the system of 12 the invention and the carrier component comprises at least 50% of the total amount of growth 13 factor(s) delivered by the system.
14 [0089] Assays to measure BMP activity [0090] Assays to characterize in vitro and in vivo function of recombinant BMPs are well 16 known in the art, (see, e.g., U.S. Patent No. 4,761,471; U.S. Patent No.
4,789,732; U.S. Patent 17 No. 4,795,804; U.S. Patent No. 4,877,864; U.S. Patent No. 5,013,649;
U.S. Patent No.
18 5,166,058; U. S. Patent No. 5,618,924; U.S. Patent No. 5,631,142; U.S.
Patent No 6,150,328;
19 U.S. Patent No. 6,593,109; Clokie and Urist, Plast. Reconstr. Surg.
2000; 105:628-637; Kirsch et al., EMBO J 2000; 19:3314-3324; Vallejo et al., J. Biotech. 2002; 94:185-194; Peel et al., J.
21 Craniofacial. Surg. 2003; 14:284-291; and Hu et al., Growth Factors, 2004; 22:29-33).
22 [0091] Such assays include: in vivo assays to quantify osteoinductive activity of a BMP
23 following implantation (e.g., into hindquarter muscle or thoracic area) into a rodent (e.g. a rat or 24 a mouse) (see, for example, U.S. Patent No. 4,761,471; U.S. Patent No.
4,789,732; U.S. Patent No. 4,795,804; U.S. Patent No. 4,877,864; U.S. Patent No. 5,013,649; U.S.
Patent No.
26 5,166,058; U. S. Patent No. 5,618,924; U.S. Patent No. 5,631,142; U.S.
Patent No 6,150,328;
27 U.S. Patent No. 6,503,109; Kawai and Urist., Olin. Orthop. Relat. Res., 1988; 222:262-267;
28 Clokie and Urist, Plast. Reconstr. Surg., 2000;105:628-637; and Hu et al., Growth Factors, 29 2004;22:29-33); in vivo assays to quantify activity of a BMP to regenerate skull trephine defects in mammals (e.g., rats, dogs, or monkeys) (see, for example, U.S. Patent No.
4,761,471 and 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 U.S. Patent No. 4,789,732); in vitro assays to quantify activity of a BMP
to induce proliferation of 2 in vitro cultured cartilage cells (see, for example, U.S. Patent No.
4,795,804); in vitro assays to 3 quantify activity of a BMP to induce alkaline phosphatase activity in in vitro cultured muscle cells 4 (e.g., 02C12 cells, ATCC Number CRL-1772) or bone marrow stromal cells (e.g., murine W-20 cells, ATCC Number CRL-2623) (see, for example, U.S. Patent No. 6,593,109;
Ruppert et al., 6 Eur J Biochem 1996;237:295-302; Kirsch et al., EMBO J, 2000;19:3314-3324;
Vallejo et al., J
7 Biotech, 2002;94:185-194; Peel et al., J Craniofacial Surg., 2003;14:284-291; and Hu et al., 8 Growth Factors, 2004;22:29-33); in vitro assays to quantify activity of a BMP to induce FGF-9 receptor 2 (FGFR3) expression in cultured mesenchymal progenitor cell lines (e.g., murine C3H10T1-2 cells) (see, for example, Vallejo et al. J Biotech 2002;94:185-194);
in vitro assays to 11 quantify activity of a BMP to induce proteoglycan synthesis in chicken limb bud cells (see, for 12 example, Ruppert et al., Eur J Biochem 1996;237:295-302); and in vitro assays to quantify 13 activity of a BMP to induce osteocalcin treatment in bone marrow stromal cells (e.g., murine W-14 20 cells; ATCC Number CRL-2623) (see, for example, U.S. Patent No.
6,593,109).
[0092] Assays to measure BMP binding and release 16 [0093] Various assays can be used to measure binding and release of recombinant BMP
17 from a carrier. For example, the amount of recombinant BMP protein can be quantified by any 18 of the techniques well known in the art, including dot blots, immunoassay (e.g., enzyme linked 19 immunosorbent assays, ELISA), measurement of the increase in radioactivity present in the release buffer when the bioimplant incorporates radiolabeled BMP and chromatography (e.g., 21 high pressure liquid chromatography, HPLC and ion-exchange chromatography).
22 [0094] Such methods are well known in the art (See for example, Harlow and Lane, Using 23 Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press.
1999; Gosling, ed., 24 Immunoassays: A Practical Approach, Oxford University Press. 2000;
Oliver, ed., HPLC of Macromolecules: A Practical Approach., Oxford University Press, 1998; Millner, ed., High 26 Resolution Chromatography: A Practical Approach. Oxford University Press, 1999; Hockfield et 27 al., Selected Methods for Antibody and Nucleic Acid Probes. Cold Spring Harbor Laboratory 28 Press. 1993; Gore, ed., Spectrophotometry and Spectrofluorimetry: A
Practical Approach.
29 Oxford University Press, 2000).

23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 [0095] For example, protocols for radioimmunoassay analysis of BMP
proteins have been 2 described (see, for example, U.S. Patent No. 4,857,456). For example, protocols for 3 immunoblot analysis of BMP proteins have been described (see, for example, Wang et al. Proc 4 Natl Acad Sci USA 1990; 87:2220-2224). For example, ELISA kits for the quantitation of protein levels of human, rat, or mouse BMP-2 are commercially available, for example, from R&D
6 Systems (catalog #DBP200, PDBP200, or SBP200). For example, ELISA kits for the 7 quantitation of protein levels of human BMP-7 are commercially available, for example, from 8 R&D Systems (catalog #DY354 or DY354E).
9 [0096] Kits [0097] In one aspect, the invention provides a kit for containing the system described 11 herein. In one embodiment, the kit comprises the necessary components for making the 12 delivery vehicle and the carrier as well as the needed growth factors.
That is, the kit of the 13 invention would comprise the necessary components for making the delivery vehicle and the 14 carrier as well as least one growth factor that is associated with, or subsequently will become associated with, the delivery vehicle and at least one growth factor associated with the carrier.
16 [0098] The kit preferably comprises a container comprising the carrier onto which may be 17 loaded or coated the associated growth factor(s).
18 [0099] Preferably, the delivery vehicle and any associated growth factor(s) are maintained 19 in separate containers, that can be combined at the time of use. This would be particularly preferable in cases where the delivery vehicle may comprise a liquid or a gel.
In such case, 21 where the delivery vehicle comprises both associated growth factor(s) and a liquid or gel, the 22 associated growth factor(s) may be kept in a separate container as a lyophilized powder. At the 23 time of use, the growth factor(s), in powder form, may be combined with the liquid or gel delivery 24 vehicle.
[00100] In a preferred embodiment, the kit of the invention would comprise at least three 26 containers for each of the following: 1) the delivery vehicle component;
2) the at least one first 27 growth factor (i.e. the growth factor(s) associated with the delivery vehicle); and, 3) the carrier 28 and the least one second growth factor (i.e. the growth factor(s) associated with the carrier). In 29 use, the at least one first growth factor, in powder form, is combined with the liquid or gel form 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 delivery vehicle and the mixture is then applied to the carrier onto which the at least one second 2 growth factor was pre-loaded.
3 [00101] In another preferred embodiment, the kit of the invention would comprise at least two 4 containers each comprising of the following: 1) the delivery vehicle component; and 2) the calcium sulphate carrier and the least one growth factor associated with the carrier and other 6 growth factor that is not associated with the carrier. In use, the liquid or gel form delivery 7 vehicle is applied to the carrier and associated GF (bound or loaded GF) and to the growth 8 factor that is not associated with the carrier ("free" GF). The free GF
then becomes incorporated 9 into the delivery vehicle.
[00102] In one preferred embodiment, the carrier is comprised of a mixture of calcium 11 sulphate and calcium phosphate particles. In particularly preferred embodiments of the present 12 invention, the carrier is comprised of a mixture of calcium sulphate and calcium phosphate 13 particles in a ratio of about 1:1 or 2:1. Preferably the carrier is coated with the at least one 14 growth factor.
[00103] In yet another preferred embodiment, the kit of the invention would comprise at least 16 three containers for each of the following: 1) the delivery vehicle component; 2) the at least one 17 first growth factor (i.e. the growth factor(s) associated with the delivery vehicle); and, 3) the 18 calcium sulphate carrier and the least one second growth factor (i.e.
the growth factor(s) 19 associated with the carrier). In use, the at least one first growth factor, in powder form, is combined with the liquid or gel form delivery vehicle and the mixture is then applied to the 21 carrier onto which the at least one second growth factor was pre-loaded.
22 [00104] In one preferred embodiment, the carrier is comprised of a mixture of calcium 23 sulphate and calcium phosphate particles. In particularly preferred embodiments of the present 24 invention, the carrier is comprised of a mixture of calcium sulphate and calcium phosphate particles in a ratio of about 1:1 or 2:1. Preferably the carrier is coated with the at least one 26 growth factor.
27 [00105] In one aspect, the kit of the invention may comprise any necessary reagents and/or 28 instruments and/or instructions and/or vessels as may be needed.
29 [00106] EXAMPLES
23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 [00107] The present invention will now be described by means of the following examples.
2 These examples illustrate the novel findings by the inventors that a multiphasic release profile of 3 a growth factor, such as rhBMP-2 produced by loading part of the BMP
within a carrier that 4 releases BMP with a sustained release and part of the BMP within a delivery vehicle that releases BMP with an initial release is more effective than carriers that only produce a burst 6 release or a sustained release. These examples also illustrate that calcium sulphate dihydrate 7 or a mixture of calcium sulphate dihydrate and calcium phosphate can be used as a carrier of a 8 growth factor, such as BMP, in improved systems, methods and compositions for increasing the 9 potency of the bioimplant.
[00108] As will be obvious to one skilled in the art it is possible to place one growth factor 11 within the carrier and a different growth factor within the delivery vehicle, resulting in different 12 release profiles of each growth factor.
13 [00109] It will be understood that the examples provided herein are intended solely to 14 illustrate the present invention and not to limit the scope of the invention in any way. Likewise, the invention is not limited to any particular preferred embodiments described herein. Indeed, 16 many modifications and variations of the invention may be apparent to those skilled in the art 17 upon reading the present specification. The invention is therefore to be limited only by the 18 terms of the appended claims, along with the full scope of equivalents to which the claims are 19 entitled.
[00110] EXAMPLE 1: Manufacture of a sustained release composite carrier containing 21 BMP by encapsulation in PLGA.
22 [00111] This example demonstrates how to form a carrier containing rhBMP-2 and which 23 releases the growth factor in a sustained release profile.
24 [00112] Materials and Methods [00113] PLGA 75/25 with inherent viscosity of 1.33 dL/g (MW = 205,000-210,000) was 26 purchased from Birmingham Polymers Inc. (Birmingham, AL). Tetracalcium phosphate (TTCP) 27 was obtained from Taihei Chemical Industrial Co. (Osaka, Japan) and dicalcium phosphate 28 anhydrous (DCPA) and dimethyl sulfoxide (DMSO) were obtained from Sigma Chemical Co.

23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 (MO, USA). Sugar particles were purchased from Tate & Lyle North America Inc. (Toronto, 2 Canada).
3 [00114] Resorbable calcium phosphate particles were prepared by mixing equimolar TTCP
4 and DCPA with deionized distilled water (ddH20) at 100% relative humidity for 24 h. The reactant was ground and sieved through 45 pm sieve.
6 [00115] Recombinant human BMP-2 (rhBMP-2, Induce Biologics Inc) in was prepared in 7 formulation buffer (1.5mg/ml, pH 4.5; 5 mm glutamic acid, 2.5% glycine, 0.5% sucrose and 8 0.01% Tween TM 80 with ddH20). The protein solution was added to vials containing CaP
9 powder and agitated for at least 15 minutes. The powder was then frozen and lyophilized.
[00116] Particles with (CaP-BMP) or without (CaP) BMP were then used to make CaP
11 particulate-PLGA scaffold blocks by phase-inversion/particle leaching as follows: PLGA was 12 dissolved in DMSO at a concentration of 11.5% (w/v). To this solution, the CaP and CaP-BMP
13 particles were thoroughly mixed according to a CaP/PLGA ratio of 2:1 (w/w). Sugar crystals 14 with size ranges of 0.85-1.18mm were dispersed in the CaP/PLGA and the mixture was solidified at -18 C in a mold. The PLGA was precipitated and the sugar crystals leached out by 16 soaking in three changes of ddH20.
17 [00117] A layer of hydroxyapatite was deposited onto and throughout the macroporous 18 composite scaffolds as follows: dry PLGA/CaP cylinders, measuring 2mm in diameter and 2mm 19 in length, were pre-wetted in 70% ethanol and immersed in 60 ml of 3xSBF
for a period of 9 days at 37 C. SBF was prepared as follows: to 1.8L of ddH20 under vigorous stirring the 21 following salts were added sequentially 29.711g NaCI, 2.206g CaCl2-2H20, 10nnl 1M HCI, 0.852 22 Na2HPO4. The final volume was brought to 2L. The SBF solution was changed daily.
23 Following coating, the 3PCC samples were rinsed in ddH20 and air dried.
24 [00118] This resulted in the formation of a macroporous composite carrier (3PS) that is able to release rhBMP-2 with a sustained release profile over at least seven days.
These results are 26 illustrated in Figure 1.
27 [00119] EXAMPLE 2: Manufacture of a sustained release carrier containing BMP by 28 encapsulation in a calcium phosphate cement 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 [00120] The present example demonstrates how to form a calcium phosphate cement (CPC) 2 carrier containing rhBMP-2 that has a sustained release profile.
3 [00121] Materials and Methods 4 [00122] Tetracalcium phosphate (TTCP) was obtained from Taihei Chemical Industrial Co.
(Osaka, Japan) and dicalcium phosphate anhydrous (DCPA) was obtained from Sigma 6 Chemical Co. Macroporous biphasic calcium phosphate granules (Eclipse) were purchased 7 from Citagenix (Laval Qc, Canada). Recombinant human BMP-2 (rhBMP-2, Induce Biologics 8 Inc) was prepared in formulation buffer (1.5mg/ml, pH 4.5; 5 mm glutamic acid, 2.5% glycine, 9 0.5% sucrose and 0.01% TweenTm 80 with ddH20).
[00123] Resorbable calcium phosphate cement particles were prepared by mixing equimolar 11 TTCP and DCPA with rhBMP-2 solution. The reactant was ground and sieved through a 300 12 and 100 pm sieve and particles between 100 and 300pm, retained.
13 [00124] This resulted in the formation of calcium phosphate cement carrier particles into 14 which the rhBMP-2 was incorporated. Upon implantation into an animal BMP
is released in a sustained manner over a period of at least several weeks.
16 [00125] To produce a CPC based sustained release carrier that also acted as a 17 macroporous carrier CPC particles (0.1 to 0.3mm) were mixed macroporous calcium phosphate 18 granules (1-2mm) in a 1:1 ratio (w/w).
19 [00126] EXAMPLE 3: Manufacture of a sustained release carrier containing BMP by use of a coating that binds BMP.
21 [00127] The present example demonstrates how to form a carrier that has a sustained 22 release profile by applying a BMP binding coating. One such method is to coat a carrier with an 23 antibody or BMP binding protein as described in our co-pending application number US
24 Application No. 13/002,444.
[00128] Materials and Methods 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 [00129] Purified polyclonal rabbit anti-human BMP-2 antibodies were purchased from Cell 2 Sciences, (Canton MA., Cat #PA0025). Macroporous biphasic calcium phosphate (BCP) 3 granules (Eclipse) were purchased from Citagenix (Laval, Qc, Canada.) 4 [00130] Sterile BCP granules were weighed out in a biosafety cabinet and placed in sterile TPP tubes (Mandel Scientific, Guelph ON, Canada). The antibody solution was diluted in 6 phosphate buffered saline to final concentration of 150, 300 and 600ng of antibody in lml PBS, 7 filter sterilized and applied to the carrier at a 1:1 v/v ratio. The samples were agitated for at 8 least 15 minutes at room temperature, before being frozen and lyophilized. BMP solution was 9 then applied to the granules, allowed to soak for 15 minutes at room temperature and then frozen and re-lyophilized.
11 [00131] This resulted in the formation of a BCP granules coated with antibody that bound 12 and slowly released the rhBMP-2 in a sustained fashion.
13 [00132] The amount of rhBMP-2 that can be bound can be increased by increasing the 14 amount of antibody used. The rate of release can be increased by using antibodies with lower affinity or avidity.
16 [00133] EXAMPLE 4: Production of a BMP containing delivery vehicle using 17 [00134] The present example demonstrates how to prepare a delivery vehicle containing 18 rhBMP-2 using F127.
19 [00135] Materials and Methods [00136] Poloxamer was prepared as follows: 100m1 of distilled water was chilled to 4 C and 21 various amounts of poloxamer 407 were added slowly with stirring over a period of several 22 hours, until all the solid prill was dissolved making a final solution ranging between 12 and 33%.
23 The poloxamer solution was then sterilized in an autoclave (121 C, 20 minutes, 30p5i).
24 Following sterilization, the poloxamer solution was kept at 4 C until use.
[00137] Lyophilized recombinant human BMP-2 powder (rhBMP-2, Induce Biologics Inc) was 26 added to the poloxamer solution and was slowly mixed.

23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 [00138] Alternatively rhBMP-2 was added from solution (1mg/ml, pH
4.5; 5 mm glutamic 2 acid, 2.5% glycine, 0.5% sucrose and 0.01% Tween 80) at a 1/10 or 1/20 ratio (v/v).
3 [00139] This resulted in the formation of a delivery vehicle that released more than 80% of 4 the rhBMP -2 over the first two days (as illustrated in Figure 2).
[00140] EXAMPLE 5: Production of a bioimplant with a multiphasic release profile 6 [00141] The present example demonstrates how to form a 3PS-F127 bioimplant containing 7 rhBMP-2 that releases the rhBMP-2 with a multiphasic release profile.
8 [00142] Materials and Methods 9 [00143] The 3PS carrier (as described in Example 1) containing 0,4.55 or 9.1pg of rhBMP-2 per 5mg of carrier was prepared and stored in Eppendorf tubes. A delivery vehicle containing 0, 11 4.55 or 9.1pg of rhBMP-2 in 45.5p1 F127 (prepared as described in Example 4) was stored in 12 Eppendorf tubes at 4 C. Immediately prior to use, the F127 was pipetted onto the 3PS carrier 13 and the carrier was mixed into the delivery vehicle.
14 [00144] This 3PS-F127 bioimplant was then used to measure BMP release in vitro and bone formation activity in vivo as described below.
16 [00145] The ratios of carrier to delivery vehicle can be varied to produce gel (1:1 ratio v:v) or 17 putties (2:1 ratio v:v). Further the ratio of BMP to carrier or the particle size of the carrier can be 18 varied to alter the sustained release profile. Finally the amount of rhBMP-2 in the carrier and 19 the delivery vehicle can be varied to alter the amount of rhBMP-2 released initially over the first few hours compared to amount released over the following weeks.
21 [00146] EXAMPLE 6: An in vitro assay for release of BMPs from bioimplants.
22 [00147] The present example describes how to measure the release of rhBMP-2 from the 23 various bioimplants described in Examples 1 to 5.
24 [00148] Materials & Methods 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 [00149] Bioimplants containing known amounts of rhBMP-2 prepared as in Examples 1 to 5 2 were transferred to Eppendorf tubes. The total amount of rhBMP-2 used was 9.1pg of rhBMP-2 3 per 5mg of carrier and 45.5p1 of F127, or 20pg of rhBMP-2 to 10mg of carrier to 100p1of F127.
4 [00150] Samples were then incubated under agitation with a 1 ml solution of release buffer comprising phosphate buffered saline (PBS) + 1% BSA at 37 C. The buffer was removed and 6 replaced with fresh release buffer after various times (e.g. 1, 2, 5, 7 and 10 days) and the 7 collected solutions were stored with 1.5 ml vials at -20 C for further analysis.
8 [00151] The amount of BMP-2 released into the buffer was measured using a commercial 9 ELISA (Quantikine TM hBMP-2 ELISA, RnD Systems). The ELISA was carried out according to the manufacturer's instructions.
11 [00152] Results 12 [00153] No BMP was detectable in release buffer collected from any of the bioimplants which 13 had not been loaded with BMP. The carrier samples which had been loaded with rhBMP-2 14 demonstrated a sustained release of rhBMP-2 over the period of the study, while samples in the delivery vehicle alone were released in an "initial release profile".
16 [00154] When the carrier and delivery vehicle were combined, various release profiles were 17 obtained depending on which component the BMP was loaded into. When 100%
of the rhBMP-18 2 (9.1pg) was loaded within the 3PS (5mg) carrier which was then mixed with 33% F127 19 (45.5p1), the BMP release profile matched the sustained pattern, where the amount of BMP
released over the first 2 days was 5ng, between days 3 and 5 it was 8ng and between days 5 21 and 7 it was lOng (Figure 3; 100-0).
22 [00155] When 100% of the rhBMP-2 (9.1pg) was loaded within 33% F127 (45.5p1) and then 23 was then mixed with the 3PS carrier (5mg) which had no BMP within it, the BMP was released 24 where the amount of BMP released over the first 1 day was 2363ng, over the second day was 381ng and then 12ng on the third day (Figure 3; 0-100).
26 [00156] When the BMP was distributed between the carrier and the delivery vehicle the 27 bioimplant demonstrated a biphasic release profile, with an intermediate initial release followed 28 by sustained release of BMP (Figure 3; 50-50).

23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 [00157] EXAMPLE 7: An in vitro assay to test the activity of released BMPs.
2 [00158] The present example describes how to determine whether the rhBMP-2 released 3 from the bioimplants retains its activity. To demonstrate that the released rhBMP is biologically 4 active, responsive cells can be cultured in with the releasate and their response to the growth factor measured. Such assays are known in the art (see Peel et al., J.
Craniofac. Surg. 2003, 6 14:284-291).
7 [00159] Materials & Methods 8 [00160] Materials with or without rhBMP-2 as described in Examples 1 to 5 were prepared.
9 Releasates were prepared as described in Example 3 except the buffer was alpha minimal essential medium with 15% fetal bovine serum and antibiotics (aMEM+15%FBS+AB) 11 [00161] 02C12 cells were seeded into 24 well tissue culture plates at 0.5x105 cells/ml, 1m1 12 alpha MEM+15%FBS per well. After various periods between 24 and 72 hours the media was 13 removed and the various releasates were applied. Negative controls included 02012 cells 14 cultured with fresh aMEM+15%FBS+AB. Positive controls included C2C12 cells incubated with aMEM+15%FlE3S+AB containing 25,50 and 10Ong/m1rhBMP-2. After 48 hours the cells were 16 lysed in 1 ml cell lysis buffer (Cellytic Sigma Aldrich) and the alkaline phosphatase (ALP) activity 17 of the cell lysates measured using the para-nitrophenol phosphate assay (Sigma Aldrich). The 18 cell protein content of the lysates was measured using Coomassie Plus Reagent (Fisher) and 19 was used to normalize ALP activity to the number of cells in each well.
[00162] Generally, to determine whether there has been any loss in activity of the BMP when 21 associated with the carrier or delivery vehicle, a standard activity curve of ALP/PTN results for 22 rhBMP-2 standards which have not been associated with the carrier or delivery vehicle is 23 determined. The concentration of active rhBMP-2 in the releasates can be determined from this 24 standard curve and this is expressed as a percentage of the total the amount of rhBMP-2 present in the releasates as determined by ELISA.
26 [00163] EXAMPLE 8: Evaluation of osteoinductive activity of multiphasic BMP
27 implants.
28 [00164] The present example describes how to determine the osteoinductive activity of BMP
29 containing bioimplants in vivo. To evaluate the ability of bioimplants to induce bone formation 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 the mouse muscle pouch assay was used. In this model the bioimplant is placed in a muscle 2 pouch made in the hind limbs of the mouse and the size of the induced bone formed is 3 proportional to the amount of BMP tested. Such assays are known in the art (see for example 4 Barr et at., Oral Surg. Oral Med. Oral Pathol. Oral Radio]. Endod., 2010;
109:531-40.) [00165] Materials and Methods 6 [00166] Bioimplants were prepared as described in Examples 1 and 5.
Under anesthesia 7 bilateral pouches were made in the thigh muscles of the hind limbs of male CD-1 mice aged 37-8 42 days, by blunt dissection. The bioimplants were then placed into sterile gelatin capsules 9 which had been placed into the muscle pouch. The muscle was pulled together and the skin closed with Mitchel clips.
11 [00167] The animals were euthanized on post-op day 28. The hind limbs were harvested 12 and fixed with 10% buffered formalin. Following fixation, the specimens were imaged using a 13 microCT scanner (General Electric Healthcare eXplore TM Locus SP).
Samples were scanned 14 and reconstructed using the manufactures software at a resolution of 59pm. Following image reconstruction, a region of interest (ROI) was determined. This area encompassed all areas 16 containing the bioimplant induced bone. These can be easily distinguished from the skeletal 17 bones based on location and density.
18 [00168] In order to analyze the quantity and quality of bone within the ROI, the voxels of the 19 mCT images were segmented into bone and non-bone phases. Segmentation was achieved by determining a threshold value for the voxel grayscale at which the voxel was counted as bone.
21 The total volume (TV), bone volume (BV), mineral density of the total volume (TV-MD), mineral 22 density of the bone volume (BV-MD), mineral content of the total volume (TV-MC), mineral 23 content of the bone volume (BV-MC) and bone volume fraction (BVF) of the ROI were 24 determined for each sample. Values were adjusted for the presence of calcium due to the carrier by using an upper threshold value that selected only carrier and subtracting it from the 26 values obtained using a lower threshold which included carrier plus new bone (see Humber et 27 at., Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology. 2010.
28 109:372-384).
29 [00169] Following completion of the microCT analysis, the specimens were either embedded in spurs resin or decalcified in formic acid and embedded in wax. Resin embedded samples 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 were evaluated by backscatter SEM while wax embedded samples were cut and stained with 2 hematoxylin and eosin (H&E) and examined by light microscopy to evaluate the tissue types 3 present at the implantation site.
4 [00170] Results [00171] A carrier and a delivery vehicle were combined as described in Example 5.
6 [00172] MicroCT analysis showed that bioimplants with all of the BMP within the 3PS carrier, 7 which had a sustained BMP release profile, produced the smallest ossicles of bone (Figure 4;
8 100-0), bioimplants with all of the BMP within the F127 delivery vehicle, which had a burst BMP
9 release profile produced intermediate sized ossicles (Figure 4; 0-100), while bioimplants with 50% of the BMP loaded into the carrier and 50% loaded into the delivery vehicle, which had a 11 multiphasic BMP release profile, produced the largest ossicles of bone (Figure 4; 50-50).
12 [00173] Backscatter SEM showed that by 28 days bone formed throughout the bioimplant 13 and onto the calcium phosphate particulate that had been incorporated into the PLGA (Figure 14 5). Histology confirmed the tissue formed was bone (Figure 6).
[00174] EXAMPLE 9: An in vivo assay for release of BMPs from bioimplants.
16 [00175] The present example describes how to measure the release of rhBMP-2 from the 17 various bioimplants described in Examples 1, 2, 3, 4 or 5 following implantation into an animal.
18 Methods to do this are well known in the art. For example see Uludag etal. J Biomed Mater 19 Res, 46, 193-202, 1999.
[00176] Materials & Methods 21 [00177] Recombinant hBMP-2 is radiolabeled with 1odine125 (1-125) by Perkin Elmer. The 22 radiolabelled rhBMP-2 (hot) is mixed with unlabeled rhBMP-2 (cold) to produce a hot cold 23 mixture of 1:100.
24 [00178] Bioimplants containing known amounts of rhBMP-2 are prepared as in Examples 1 to 5. These bioimplants are then implanted into animals as described in Example 8. At various 26 times the animals are sacrificed and the implant site is dissected out.
The dissected tissue is 27 then weighed, and the amount of radioactivity determined using a gamma counter.

23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 [00179] To determine whether the counts are associated with protein, the tissue is 2 homogenized in 0.5m1 PBS+0.5 /0 BSA. Two mls of ice cold 10%
trichloroacetic acid are added 3 to the homogenate and is then held for at least 1 hour at 4 C. The homogenate is then 4 centrifuged and the supernatant removed. The radioactivity of the precipitate is then measured using a gamma counter.
6 [00180] The radioactivity associated with implants is corrected for the decay and the total 7 amount of BMP remaining in the implant is estimated.
8 [00181] EXAMPLE 10: Production of a carrier with a short sustained release profile.
9 [00182] The present example describes means of producing a carrier that releases a growth factor with a short sustained release profile.
11 [00183] Materials & Methods 12 [00184] Macroporous biphasic calcium phosphate (BCP) granules (Eclipse) were purchased 13 from Citagenix (Laval, Qc, Canada.) Recombinant human BMP-2 (rhBMP-2, Induce Biologics 14 Inc) was prepared in formulation buffer (1.5mg/ml, pH 4.5; 5 mm glutamic acid, 2.5% glycine, 0.5% sucrose and 0.01% Tween TM 80 with ddH20).
16 [00185] Sterile rhBMP-2 solution was incubated with sterile BCP
granules at a ratio of 9.1pg 17 per 5mg or 4.55pg per 5mg (BMP per BCP) for 15 minutes under shaking.
The samples were 18 then frozen and lyophilized aseptically.
19 [00186] Following lyophilization the carriers were weighed into 5mg aliquots and placed in sterile Eppendorf tubes. Some tubes had 33% F127 (45.5p1 added). The BMP
release profile 21 was then determined as described in Example 6.
22 [00187] Results 23 [00188] Carriers that were not coated with F127 (BCP) showed a burst release profile with 24 the largest amount of BMP released over the first day and then decreasing amounts of BMP
released at each subsequent time point. Mixing the BCP within the F127 (BCP-Pol) resulted in a 26 short sustained release profile where similar amount of BMP were collected each day over the 27 first 4 days (Figure 7).
23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 [00189] EXAMPLE 11: Altering the sustained release profile of the carrier.
2 [00190] The present example describes a means of altering the release profile from a carrier.
3 [00191] Materials & Methods 4 [00192] PLGA with differing inherent viscosities and molecular weights were purchased from Birmingham Polymers Inc. (Birmingham, AL). Carriers were then made using these PLGAs as 6 described in Example 1. The BMP release profile from these carriers was determine according 7 to the method of Example 6.
8 [00193] Results 9 [00194] All carriers produced sustained release profiles. However the amount of BMP
released differed depending on the viscosity/molecular weight of the PLGA
used. The carriers 11 made with low viscosity PLGA (P01-1) released more rhBMP-2 than those using the high 12 viscosity (P01-2) PLGA over the 12 week duration of the study (Figure 8).
13 [00195] EXAMPLE 12: Altering bound and unbound protein distribution during 14 Ivophilization.
[00196] The present example describes a means of altering the distribution of a protein 16 between bound to the carrier particles and unbound lyophilisate by varying the volume of 17 solution lyophilized but keeping total protein and carrier content fixed. This means allows for 18 distribution of protein between carrier-associated and delivery vehicle-associated protein if a 19 delivery vehicle is subsequently added to the lyophilization container.
[00197] Materials and Methods 21 [00198] Experimental Design: To test the effect that varying the volume of protein buffer 22 added to the carrier prior to lyophilisation has on the distribution of the lyophilized material, the 23 carrier to liquid protein volume ratio was varied and the total amount of protein (bovine serum 24 albumin, "BSA'') and carrier were fixed at 1mg and 400mg, respectively.
[00199] Based on the criteria above the study was designed as set forth in Table 1, wherein 26 the volume of protein solution added (i.e., 2.0, 1.5, 1.0, 0.75 and 0.5 ml/vial) and the 27 concentration of protein added (i.e., 0.5, 0.67, 1.0, 1.33 and 2.0 mg/ml) were varied.

23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 [00200] Table 1: Experimental design.
Gp Carrier Volume of Protein solution added (ml/vial) mg/vial By BSA concentration (mg/m1) n 2 1.5 1.0 0.75 0.5 1 400 0.5 3 2 400 0.67 3 3 400 1.0 3 4 400 1.33 3 400 2.0 3 3 [00201]
Sample preparation: BSA was prepared in formulation buffer (FB) at 2mg/m1;
4 1.5mg/m1; 1mg/ml; 0.75mg/m10.5mg/m and Omg/ml. 400mg of carrier was put in each vial 5 containing BSA and FB. Various volumes of protein solution were placed in each vial at the 6 ratios provided in Table 1 and the vials were held at room temperature for 30 minutes. Vials 7 were then frozen and lyophilized. Following lyophilisation, each vial was examined and the 8 appearance of the protein lyophilisate was categorized and photographed (FIG. 9 A-C).
9 [00202] Scoring: Distribution of protein lyophilisate was scored between 0 and 4, with 0 representing no clear lyophilisate particles visible and 4 representing a clear separation of 11 carrier and protein with a sheet of protein lyophilisate visible.
12 [00203] Protein measurements: To quantitate the amount of protein bound to the carrier and 13 the amount lyophilized separately from the granules the lyophilized materials were transferred 14 from the vial to a centrifuge tube and 1 ml of PBS was added to the centrifuge tube and to the vial. The containers were vortexed and rinse solution was collected and centrifuged. The 16 supernatants were assayed for protein content using the Coomassie-Plus protein assay 17 according to the manufacturer's instructions. The amount of bound protein was calculated by 18 subtracting the amounts of protein released from granules and the vial from the amount of 19 protein loaded (1000pg).
[00204] Results 21 [00205] There was a significant difference in distribution of lyophilisate between group 1, 22 which had the lowest protein concentration (i.e., 0.5 mg/ml) and groups 3(1.0 mg/ml), 4(1.33 23 mg/ml) and 5 (2.0 mg/m1)(Table 2). The samples in group 1 had some protein lyophilisate 24 visible on the walls of the vial (FIG. 9A). No lyophilisate was visible between the carrier 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 particles in group 1 samples. In the group 3 samples, some protein lyophilisate was visible on 2 the glass walls of the vial. However, large chunks of protein lyophilisate resembling white or 3 translucent snowflakes were also visible between the carrier particles in group 3 samples (FIG.
4 9B). In group 5 samples, distinct rims of protein formed on the surface of the glass vial (FIG.
9C). These rims of protein projected chunks of protein lyophilisate. Chunks were also seen lying 6 between the carrier particles in group 5 samples.
7 [00206] Table 2: Distribution of protein lyophilisate bound to granules and unbound.
Gp Carrier Volume of Protein solution added (ml/vial) mg/vial By BSA concentration (mg/ml) Score ___________________ 2 1.5 1.0 0.75 0.5 400 0.5 1.0 0.0 2 400 0.67 1.7 0.3 3 400 1.0 2.2 0.8 4 400 1.33 2.3 0.8 5 400 2.0 3.0 0.5 8 ANOVA on RANKS P = 0.038 9 [00207] Group 1 vs Group 5, Group 1 vs Group 4 and Group 1 vs Group 3 were all significantly different (Table 2).
11 [00208] Measurement of bound protein indicated there were significant differences between 12 the amounts of bound protein based on the volume of solution added prior to lyophilization.
13 Specifically, the amount of bound protein changed from 68% to 39% when the volume of 14 solution used to deliver 1mg of protein was changed from 0.5ml to 2m1 per 400mg of carrier (Table 3, see bound protein in groups 1 and 5, respectively).
16 [00209] Table 3: Protein measurements G Releged,frbk:
Granules Via 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 ANOVA P=0.019 P = 0.023 P<0.001 1 *Bound was calculated by subtracting the amounts of protein released from granules and the 2 vial from the amount loaded (1000pg).

4 [00210] Post Hoc analysis of the bound protein group indicated that there were significant 5 differences between: group 1 and groups 3, 4, and 5; group 2 and groups 4 and 5; and group 3 6 and group 5 (Table 4).
7 [00211] Table 4: Comparisons for bound protein.
Comparisons for factor:
Comparison Diff of t Unadjusted P Critical Level Significant?
Means Col 1 vs. Col 5 292.423 8.666 0.00000581 0.005 Yes Col 1 vs. Col 4 258.966 7.674 0.0000169 0.006 Yes Col 2 vs. Col 5 204.304 6.054 0.000123 0.006 Yes Col 1 vs. Col 3 171.484 , 5.082 0.000477 0.007 Yes Col 2 vs. Col 4 170.848 5.063 0.000490 0.009 Yes Col 3 vs. Col 5 120.939 3.584 0.00498 0.010 Yes Col 1 vs. Col 2 88.119 2.611 0.0260 0.013 No Col 3 vs. Col 4 87.483 2.592 0.0268 0.017 No Col 2 vs. Col 3 83.365 2.470 0.0331 0.025 No Col 4 vs. Col 5 33.457 0.991 0.345 0.050 No 9 [00212] Example 13: Effect of varying carrier and P407 amount on BMP
release.
[00213] The present example describes a means for varying the release profile of BMP from 11 the carrier by varying the amount of delivery vehicle and carrier used.
12 [00214] Materials and Methods 13 [00215] Experimental Design: To test the effect that varying the amount of delivery vehicle 14 and carrier have on the release profile of BMP, the carrier (biphasic calcium phosphate BCP) amount and the delivery vehicle (33% P407 gel) amount were varied in bioimplants wherein the 16 amount of BMP added to the carrier particles was fixed at 40pg/sample and the BMP was 17 lyophilized onto the carrier granules. The study design is further described in Table 5.
18 [00216] Table 5: Study design.

23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 ; -s :V,- tr-14Z4Z:titieg . ;1 . ' .41friVa 2 [00217] Preparation of Materials: Sterile macroporous BCP granules (0.5-1mm diameter) 3 comprising 80% a-tricalcium phosphate, 20% Hydroxyapatite were purchased from Citagenix 4 Inc. (Laval, QC). BMP-2 (1mg/m1) was prepared by Induce Biologics Inc. A
33% poloxamer 407 (P407) gel was prepared by adding 33g of poloxamer 407 (BASF) to cold water.
The solution 6 was then sterilized by autoclaving. The poloxamer gel was kept at 2-8 C
after sterilization.
7 [00218] BMP was lyophilized onto the carriers as follows. The required amount of carrier was 8 weighed out and placed into a sterile Eppendorf tube. The desired amount of BMP-2 was added 9 to the carrier and was held at room temperature for 30 minutes prior to freezing. Once frozen the Eppendorf tubes were placed in a bench top lyophilizer and lyophilized overnight. All 11 procedures were performed aseptically to maintain sterility.
12 [00219] BMP release: The lyophilized samples were weighed and placed in Eppendorf tubes 13 to which P407 gel was added and allowed to soak for 20 minutes.
Following this, 1m1 of 14 PBS+0.1% BSA was added to each tube which was then placed on a shaker in a 37 C
incubator and gently shaken. At each collection time point (days 1, 2, 3, 4, 7) the tubes were 16 removed, centrifuged and the PBS+BSA removed and fresh PBS+BSA added.
The collected 17 PBS+BSA was then stored frozen until analysed.
18 [00220] Analytical methods: The amount of BMP released into the solutions was determined 19 using a BMP-2 ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions 21 [00221] Statistical Analysis: The data were tested for normality and equal variance. Normally 22 distributed data with equal variance was tested for significant differences using 2 Way ANOVA
23 (Carrier and P407 were used as the factors). All other data were tested using ANOVA on 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 RANKs. Post ¨ Hoc testing was performed all pairwise using the Student-Newman-Keuls 2 Method. All statistical tests were performed using Sigma Stat v3.5.
3 [00222] Results 4 [00223] The addition of P407 slowed release of BMP for up to 7 days (Table 6). This was a surprising result as P407 gels were previously reported to be effective at slowing drug release 6 for only a matter of hours, after which time it had dispersed into the buffer. The amount of P407 7 affected BMP release over the first 7 days. From day 4 the amount of P407 initially used 8 determines whether there is a difference between no P407 and + P407 groups (Table 6). The 9 amount of carrier present affected the release of BMP in the presence or absence of P407, from day 2 to 7, with increasing amount of carrier reducing the amount of BMP
released (Table 6).
11 [00224] Table 6: BMP release over seven days from bioimplants containing various amounts 12 of P407 gel and BCP particles.
Nt,.,.../Vi, %..4.'; !f4:21:4:14ipp PrP467V!-1-; ,410Mg.7601ifP:40-1411a4PkallggliilYPIPrtfil ' ' -Riov-:=1+;:;.:ro ::',,,,:a.., L= :-!:!,4'4=$17..,,,ir-.4., p:,.,..,,,,-#F,t,--;-,-;.,5,4--'s,o.,..- ,i.1-, '-lm-'0',w.v,-',- - ''L7...-,Nffikviozzo44.-,::!
:fiirc04,:;:ii l4ni4ii 0 qq11.q* I,JoOrG ,v4spvideirrovall.'y VASPIWIN .,,,, .mqVge Sittc'SJ:Wiz;
'7 1) `. - ' :1'" ''''''''''' 7 ; : ' ' : e ,"''".: :';:'= ;
.]":;'''''17':'! ¨ .4.-,,=*,:t,-,-,-41%7 ,,,,,r.,1=-=, "gio Day 1 8838 1436 5718 1309 4404 1260 9018 7135 Day 2 2923 1040 1282 330 1884 504 532 152 Day 3 1511 1043 884 156 1141 542 674 260 Day 4 332 27 443 192 298 54 253 72 Day 7 1004 10 1301 265 1085 93 1028 403 l'illVre ::'::;490ViP:11:',Vr70 :i.4.A.F.F.ipm$,Ocr.)!.1 ,4 09,ffp,:mmolAilogo-r-i#4,7ge . .
,T]Ty),Tv,;14" '.,.,-,:::,...}?,;;: !..-:,1.7,..11., 44-1070gPr:flit7õtal .'!:.1, P;i4t Day 1 4320 510 4980 672 6594 1152 12420 1701 Day 2 865 106 999 130 1362 422 320 119 Day 3 409 104 585 393 628 57 220 56 Day 4 1140 151 1164 173 1338 81 775 26 Day 7 882 201 ' 885 61 949 160 479 44 t.;=,',C . ,, = .80mg.'30 pl:P407: : ::=-f 80 g-6 01; P4p7 ' :4.-';itigiing.7120.14,F30:7 :'.43 .... -;=-= . 1 .., 7 : e . : , , , .. = = , ,- -' !::=,'-'7,::' , ,i':r.7.'....' ' Y

23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 AIRVOR'!I-Y; .171= ft,.. ___________________ - tl-, .;11APP-r-.4.9' õ..
,.., ' ..., ..:ii9j i::: = ;
Day 1 6828 2359 4908 660 ' 5388 1100 ' 2108 Day 2 768 124 862 290 1288 - 670 279.0 26 Day 3 996 113 887 90 918 111 648.8 52 Day 4 876 205 706 104 844 ' 106 413.0 96 Day 7 698 67 995 253 843 205 375.3 87 2 [00225]
Table 7: Statistical analysis of BMP release over seven days from bioimplants 3 containing various amounts of P407 gel. 2 Way ANOVA (P values).
Dy 1 Dy 2 Dy 3 Dy 4 Dy 7 Total CARRIER 0.174 <0.001 0.002 <0.001 <0.001 0.002 P407 <0.001 <0.001 0.08 <0.001 <0.001 <0.001 SCAFxP407 0.015 0.006 0.642 0.19 0.283 <0.001 [00226] Day one data showed that there was significantly more BMP released from the 6 carrier in the absence of P407 than when it was present (Table 8).
7 [00227] While the amount of carrier alone was not considered to impact BMP release on day 8 1 there was an interaction between carrier amount and P407 amount.
Specifically 20mg of 9 carrier the amount of P407 gel used significantly influenced BMP released (30 v 120 and 30 v 60) while in samples with 40 or 80mg of carrier this was not observed (Table 8).
11 [00228] Table 8: ANOVA table for day one results.
Comparisons for factor: P407 within 20mg carrier group Comparison Duff of t Unadjusted P Critical Level Significant?
Means 0.000 vs. 120.000 8718.000 7.829 , 0.000 0.009 Yes 0.000 vs. 60.000 7404.000 6.649 0.000 0.010 Yes 30.000 vs. 120.000 4434.000 3.982 0.001 0.013 Yes 0.000 vs. 30.000 4284.000 3.847 0.001 0.017 Yes 30.000 vs. 60.000 3120.000 2.802 0.010 0.025 Yes 60.000 vs. 120.000 1314.000 1.180 0.250 0.050 No Comparisons for factor: P407 within 40mg carrier group Comparison Diff of t Unadjusted P Critical Level Significant?
Means 0.000 vs. 30.000 8100.000 7.274 0.000 0.009 Yes 0.000 vs. 60.000 7440.000 6.681 0.000 0.010 Yes 0.000 vs. 120.000 5826.000 5.232 0.000 0.013 Yes 120.000 vs. 30.000 2274.000 2.042 0.052 0.017 No 120.000 vs. 60.000 1614.000 1.449 0.160 0.025 No 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 60.000 vs. 30.000 660.000 0.593 0.559 0.050 No Comparisons for factor: P407 within 80 mg carrier group Comparison ' Diff of t Unadjusted P Critical Level Significant?
Means 0.000 vs. 60.000 6330.000 5.684 0.000 0.009 Yes 0.000 vs. 120.000 5850.000 5.253 0.000 0.010 Yes 0.000 vs. 30.000 4410.000 3.960 0.001 0.013 Yes 30.000 vs. 60.000 1920.000 1.724 0.098 0.017 No 30.000 vs. 120.000 1440.000 1.293 0.208 0.025 No 120.000 vs. 60.000 480.000 0.431 0.670 0.050 No 2 [00229] Day two data showed that both carrier amount and P407 amount significantly 3 impacted the BMP release, with interactions occurring (Table 9). The amount of P407 needed 4 for effect was dependant on the amount of carrier.
[00230] Table 9: ANOVA table for day two results.
Comparisons for factor: P407 within 20 Comparison Diff of t Unadjusted P Critical Level Significant?
Means 30.000 vs. 0.000 2391.000 6.785 0.000 0.009 Yes 30.000 vs. 60.000 1640.400 4.655 0.000 0.010 Yes 120.000 vs. 0.000 1352.400 3.838 0.001 0.013 Yes 30.000 vs. 120.000 1038.600 2.947 0.007 0.017 Yes 60.000 vs. 0.000 750.600 2.130 0.044 0.025 No 120.000 vs. 60.000 601.800 1.708 0.101 0.050 No Comparisons for factor: P407 within 40 Comparison Diff of t Unadjusted P Critical Level Significant'?
Means 120.000 vs. 0.000 1042.200 2.957 0.007 0.009 Yes 60.000 vs. 0.000 679.800 1.929 0.066 0.010 No 30.000 vs. 0.000 545.400 1.548 0.135 0.013 No .
120.000 vs. 30.000 496.800 1.410 0.171 0.017 No 120.000 vs. 60.000 362.400 1.028 0.314 0.025 No 60.000 vs. 30.000 134.400 0.381 0.706 0.050 No Comparisons for factor: P407 within 80 Comparison Dift'of t Unadjusted P Critical Level Significant?
Means 120.000 vs. 0.000 1009.200 2.864 0.009 0.009 No 60.000 vs. 0.000 582.600 1.653 0.111 0.010 No 120.000 vs. 30.000 520.200 1.476 0.153 0.013 No 30.000 vs. 0.000 489.000 1.388 0.178 , 0.017 No 120.000 vs. 60.000 426.600 1.211 0.238 _ 0.025 No 60.000 vs. 30.000 93.600 0.266 0.793 _ 0.050 No Comparisons for factor: SCAF within 30 Comparison Diff of t Unadjusted P Critical Level Significant?
Means 20.000 vs. 80.000 2154.600 6.114 0.000 0.017 Yes 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 20.000 vs. 40.000 2057.400 5.838 0.000 0.025 Yes 40.000 vs. 80.000 97.200 0.276 0.785 0.050 No "1 2 [00231] Day three results indicated that the amount of carrier was the primary factor that 3 affected BMP release. However P407 gel amount neared significance in several groups (Table 4 10).
[00232] Table 10: ANOVA table for day three results Comparisons for factor: CARRIER
Comparison Diff of t Unadjusted P Critical Level Significant?
Means 20.000 vs. 40.000 592.200 3.876 0.000720 0.017 Yes 80.000 vs. 40.000 402.090 2.632 0.0146 0.025 Yes 20.000 vs. 80.000 190.110 1.244 0.225 0.050 No Comparisons for factor: P407 Comparison Diff of t Unadjusted P Critical Level Significant?
Means 30.000 vs. 0.000 457.820 2.595 0.0159 0.009 No 120.000 vs. 0.000 381.140 2.161 0.0409 0.010 No 60.000 vs. 0.000 270.840 1.535 0.138 0.013 No 30.000 vs. 60.000 186.980 1.060 0.300 0.017 No 120.000 vs. 60.000 110.300 0.625 0.538 0.025 No 30.000 vs. 120.000 76.680 0.435 0.668 0.050 No 7 [00233] Day four results indicated that both the amount of carrier and P407 gel affected BMP
8 release, although there appeared to be no interaction between the two (Table 11). There were 9 no differences in the amount of P407 gel, as long as more than 30p1 of gel had been used. BMP
release differed between all 3 amounts of carrier used (Table 11).
11 [00234] Table 11: ANOVA table for day four results.
Comparisons for factor: CARRIER
Comparison Diff of t Unadjusted P Critical Level Significant?
Means 40.000 vs. 20.000 772.380 15.479 5.465E-014 0.017 Yes 40.000 vs. 80.000 394.380 7.904 0.0000000391 0.025 Yes 80.000 vs. 20.000 378.000 7.575 0.0000000817 0.050 Yes Comparisons for factor: P407 Comparison Diff of t Unadjusted P Critical Level Significant?
Means 120.000 vs. 0.000 346.440 6.013 0.00000330 0.009 Yes 30.000 vs. 0.000 302.820 5.256 0.0000218 0.010 Yes 60.000 vs. 0.000 290.780 5.047 0.0000369 0.013 Yes 120.000 vs. 60.000 55.660 0.966 0.344 0.017 No 120.000 vs. 30.000 , 43.620 0.757 0.456 0.025 No 30.000 vs. 60.000 12.040 0.209 0.836 0.050 No 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 2 [00235] Day seven results were similar to day 4 results with both the amount of carrier 3 particles and P407 gel affecting BMP release (Table 12). At this time however a minimum 4 amount of 60p1 of P407 gel per implant must be used.
[00236] Table 12: ANOVA table for day seven results.
Comparisons for factor: CARRIER
Comparison Diff of t Unadjusted P Critical Level Significant?
Means 20.000 vs. 80.000 376.455 4.868 0.0000581 0.017 Yes 20.000 vs. 40.000 305.310 3.948 0.000601 0.025 Yes 40.000 vs. 80.000 71.145 0.920 0.367 0.050 No Comparisons for factor: P407 Comparison Diff of t Unadjusted P Critical Level Significant?
Means 60.000 vs. 0.000 432.820 4.847 0.0000613 0.009 Yes 120.000 vs. 0.000 331.420 3.712 0.00109 0.010 Yes 30.000 vs. 0.000 233.820 2.619 0.0151 0.013 No 60.000 vs. 30.000 199.000 2.229 0.0355 0.017 No 60.000 vs. 120.000 101.400 1.136 0.267 0.025 No 120.000 vs. 30.000 97.600 1.093 0.285 0.050 No 7 [00237] Discussion 8 [00238] Taken together, these results show that it is possible to vary the release profile of 9 BMP by varying the amount of P407 and carrier used. These results also show that, in contrast to previous reports of using P407 for drug delivery over a period of a few hours, the use of P407 11 gel in combination with carrier results in inhibition of protein release for up to 7 days. It is 12 contemplated herein that, after the majority of the P407 has dissolved in the first several hours, 13 a thin layer of P407 gel might remain on the surface of the carrier, slowing the rate of protein 14 release.
[00239] Example 14: Evaluation of in vitro protein release from different carrier 16 particles.
17 [00240] The present example describes bioimplants comprising calcium sulphate dehydrate 18 (CSD) particles onto which rhBMP-2 was lyophilized. These bioimplants produced a larger and 19 more consistent release of BMP over 14 days relative to bioimplants comprising 2 types of calcium phosphate particles as the carrier.
23580844.2 CA 2,868,992 Blakes Ref: 75312100030 1 [00241] Materials and Methods 2 [00242] Experimental Design: Three carriers were tested: calcium sulphate dihydrate (CSD), 3 hydroxyapatite (HAp) and biphasic calcium phosphate (BCP) in bioimplants, wherein the ratio of 4 BMP to carrier was 40pg:20mg, the ratio of carrier to delivery vehicle (i.e., P407) was 200p1:20nrig and wherein the BMP was lyophilized onto carrier granules.
Experimental design is 6 further set forth in Table 13.
7 [00243] Table 13: Carrier Comparison.

24.1V = = ;".,V,7µ 4?",.;,. A.7 rtir ' cf4 = ;
= . = = ,!t CSD(B)+P407 Calcium sulphate 4 HAp(B)+P407 Hydroxyapatite 4 BCP(B)+P407 Biphasic Calcium phosphate 4 9 [00244] Preparation of Materials: Sterile macroporous BCP granules (0.5-1mm diameter) comprising 80% 11-tricalcium phosphate, 20% Hydroxyapatite were purchased from Citagenix 11 Inc. (Laval, QC). Sterile CSD granules (0.5-1.2 mm) were prepared by grinding Osteoset 12 pellets (Wright Medical Technology Canada Ltd., Mississauga, ON) and sieving between 13 1.18mm and 0.5mm sieves. Sterile hydroxyapatite granules were obtained from Tissue 14 Regeneration Therapeutics (Toronto, ON). BMP-2 (1mg/m1) was prepared by Induce Biologics Inc. A 33% poloxamer 407 (P407) gel was prepared by adding 33g of poloxamer 407 (BASF) to 16 cold water. The solution was then sterilized by autoclaving. The poloxamer gel was kept at 2-17 8 C after sterilization.
18 [00245] BMP lyophilization onto carrier particles: The required amount of carrier was 19 weighed out and placed into a sterile Eppendorf tube. The desired amount of BMP-2 was added to the carrier and was held at room temperature for 30 minutes prior to freezing. Once frozen, 21 the Eppendorf tubes were placed in a bench top lyophilizer and lyophilized overnight. All 22 procedures were performed aseptically to maintain sterility.
23 [00246] BMP release in vitro: 80plof P407 gel was added to the carrier and associated 24 rhBMP-2 and allowed to soak for 20 minutes. Following this, 1m1 of PBS+0.1% BSA was added to each tube which was then placed on a shaker in a 37 C incubator and gently shaken. At each 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 collection time point (days 1, 2, 3, 4, 7, 10 and 14) the tubes were removed, centrifuged and the 2 PBS+BSA removed and fresh PBS+BSA added. Collected PBS+BSA was then stored frozen 3 until analysed.
4 [00247] Analytical methods: The amount of BMP released into the solutions was determined using a BMP-2 ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer's 6 instructions.
7 [00248] Statistical Analysis: The data were tested for normality and equal variance. Normally 8 distributed data with equal variance were tested for significant differences using ANOVA. All 9 other data were tested using ANOVA on RANKs. Post ¨ Hoc testing was performed all pairwise using the Student-Newman-Keuls Method. All statistical tests were performed using Sigma Stat 11 v3.5.
12 [00249] Results 13 [00250] Post-Hoc testing indicated that the calcium sulphate carrier particles released more 14 BMP than the BCP carrier particles at all time points tested (Table 14).
The CSD carrier particles also released more BMP than the Hap carrier particles at day 1, day 2 and day 7.
16 BMP release from the HAp carrier particles differed from BCP on days 7 and 10.
17 [00251] Table 14: BMP release from various carrier particles.
.1(= ' Ipalciuiii=gtilphatE rf.;'e Hydroxya haici4 -,rw4 .14;14,- = = ' r,: '= = 4-'" = = . , f 1 1863 231 138 34 673 608 0.004 2 910 171 218 88 400 165 0.003 3 1457 650 1363 289 690 421 0.181 4 1200 1381 1070 46 344* not done 7 744 124 397 73 172 50 <0.001

10 373 254 199 35 109 19 0.011 14 232 58 150 30 97 2 0.013 total 6780 2431 3536 438 2486 916 0.032 18 * only a single sample was measured, consequently the ANOVA was not performed at this time 19 point.

23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 [00252] Discussion 2 [00253] These results show that the CSD carrier particles released more BMP-2 than the 3 other carrier particles tested in total over 14 days and at all but the 3 day timepoint.
4 [00254] EXAMPLE 15: Production of a BMP carrier having improved potency in vivo relative to known carriers used in multiphasic BMP bioimplant.
6 [00255] The present example describes the evaluation of various carrier components to 7 determine which produces the most bone when used as part of a multiphasic BMP bioimplant.
8 In this example "improved bone growth" or "improved capacity for boney ossicle formation"
9 refers to an increase in the size and/or density of bone ossicles relative to that of known carriers comprising the same BMP. The results of this study show that bioimplants using CSD particles,

11 onto which rhBMP-2 was lyophilized produced larger ossicles of bone when implanted than

12 bioimplants containing hydroxyapatite or biphasic calcium phosphate carriers.

13 [00256] Materials and Methods

14 [00257] Experimental Design: To identify a carrier with relatively high bone producing capacity calcium sulphate dihydrate (CSD), hydroxyapatite (HAp) and biphasic calcium 16 phosphate (BCP) were tested in bioimplants wherein the ratio of BMP to implant volume was 17 fixes at 40pg:20mg, the ratio of carrier to delivery vehicle (i.e., P407) was fixed at 200p1:20mg 18 and the BMP was lyophilized onto carrier granules. Experimental design is further set forth in 19 Table 15.
[00258] Table 15: Carrier Comparison , t,j1'= = . = . -; = %-..1,`A.
- - , = =
Al2- =
, , CS D(B)+P407 Calcium sulphate 8 HAp(B)+P407 Hydroxyapatite 8 BCP(B)+P407 Biphasic Calcium phosphate 8 22 [00259] Preparation of Materials: Sterile macroporous BCP granules (0.5-1mm diameter) 23 comprising 80% 11-tricalcium phosphate, 20% Hydroxyapatite were purchased from Citagenix 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 Inc. (Laval, QC). Sterile CSD granules (0.5-1.2 mm) were prepared by grinding Osteoset pellets 2 (Wright Medical Technology Canada Ltd., Mississauga, ON) and sieved between 1.18mm and 3 0.5mm sieves. Sterile hydroxyapatite granules were obtained from Tissue Regeneration 4 Therapeutics (Toronto, ON). BMP-2 (1mg/m1) was prepared by Induce Biologics Inc. A 33%
poloxamer 407 (P407) gel was prepared by adding 33g of poloxamer 407 (BASF) to cold water.
6 The solution was then sterilized by autoclaving. The poloxamer gel was kept at 2-8 C after 7 sterilization. BMP was lyophilized onto the carriers as follows: the required amount of carrier 8 was weighed out and placed into a sterile Eppendorf tube. The desired amount of BMP-2 was 9 added to the carrier and was held at room temperature for 30 minutes prior to freezing. Once frozen the Eppendorf tubes were placed in a bench top lyophilizer and lyophilized overnight. All 11 procedures were performed aseptically to maintain sterility.
12 [00260] Surgical Model: The osteoinductivity of the various bioimplants was evaluated in the 13 mouse muscle pouch assay (Barr et al. Oral Surg Oral Med Oral Pathol Oral Radio! Endod.
14 2010;109(4):531-40).
[00261] Samples where poloxamer was to be mixed with carrier were prepared by pouring 16 the carrier granules onto a sterile stainless steel tray. The poloxamer was kept on ice and the 17 appropriate amount of poloxamer gel was applied by pipette to the carrier granules. The carrier 18 and gel were mixed and then carefully placed into a gelatin capsule which was then placed in 19 the muscle pouch.
[00262] Male IGS mice (approximately 22gm) had intramuscular pouches formed in their 21 biceps femoris muscle by blunt dissection. The bioimplant was then placed into the pouch. The 22 skin was then pulled together and closed using Michel clips.
23 [00263] The mice were monitored daily. Originally the mice were to be euthanized after 28 24 days. However due to some implants forming so much bone that bridging occurred between the spine and the femur, which restricted the mice's mobility, all mice were sacrificed after 18 days.
26 Following sacrifice of the animals, the rear limbs were dissected out and fixed using neutral 27 buffered formalin.
28 [00264] Analytical methods: The amount of bone formed by the bioimplants was determined 29 by micro CT. Appropriate values were adjusted for the presence of calcium from the residual carrier as previously described (Humber et al. Oral Surg. Oral Med Oral Pathol. Oral Radiol.

23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 Endod. 2010 Mar; 109(3):372-84). Briefly, the region where the implant had been placed was 2 imaged using a General Electric Healthcare eXplore TM Locus SP microCT
scanner. The residual 3 carrier and any new mass that had formed around the implant in the muscle (collectively called 4 an ossicle) was outlined every 10 slices to define the region of interest (ROI).
[00265] Carrier material was denser than the new bone. Therefor it was possible to 6 determine threshold values for new bone and carrier separately by imaging multiple samples 7 from each group and taking an average of the grey-scale values. For the purpose of 8 standardization, the lowest carrier threshold value obtained for a material (i.e., CSD) was used 9 for all carriers (i.e., 1835). Similarly, a single value for new bone was used (i.e., 555).
[00266] Analyses were performed using the 2 threshold values (i.e., 1835 and 555). The 11 upper threshold distinguished carrier from bone and soft tissue, while the lower distinguished 12 bone + carrier from soft tissue. By subtracting the upper threshold values from the lower 13 threshold values the values for bone only were determined. Seven different parameters were 14 measured using the microCT. Table 16 describes the parameters obtained directly from the microCT and any thresholding that impacted the result. Table 17 describes the derived 16 parameters and how they are calculated.
17 [00267] Table 16: Reported parameters provided by microCT.
= = ::.= ' Threshold.:
-.Parameter Abbreviation . . . Description.
Total Volume TV Total volume of ROI. Includes volume occupied by No bone, carrier and soft tissues Bone Volume By Volume occupied by yokels with grey scale above Yes (SV) threshold value in the ROI
When using the upper threshold this would represent the carrier volume When using the lower threshold this would be a measure of the bone+carrier volume Bone Mineral BMC Mineral content within the ROI. This is based on No Content comparison of greyscale of all voxels in Bone Mineral BMD BMC/TV No Density 23580844.2 CA 2,868,992 Slakes Ref: 75312/00030 Tissue TMC Mineral content of tissue within the ROI with voxels Yes Mineral (uTMC) greater than the threshold value (i.e. bone) Content When using the upper threshold this would represent the mineral content due to the carrier Tissue TMD TMC/BV Yes Mineral (uTMD) When using the upper threshold this would represent Density the mineral density of the carrier Bone Volume BVF BV/TV The fraction of the total volume occupied by Yes Fraction tissue with a grey scale greater than the threshold (SVF) value When using the upper threshold this would represent the percentage of the ossicle occupied by carrier 2 [00268] Table 17: Calculated parameters (Lower threshold-upper threshold).
Parameter. Abbreviatio . :149W-calculated:
. = = . ,rt..= . .
Dependant Adjusted Bone aBV BV-SV Yes Volume Adjusted Tissue aTMC TMC-uTMC Yes Mineral Content Adjusted Tissue aTMD aTMC/aBV Yes Mineral Density Adjusted Bone aBVF aBV/TV Yes Volume Fraction 4 [00269] The two measures used to determine osteoinductive activity were total volume (TV) and adjusted bone volume (aBV).
6 [00270] Histology: Following micro CT analysis, samples were decalcified and processed for 7 light microscopy.
8 [00271] Statistical Analysis: MicroCT parameters were tested for normality and equal 9 variance. Normally distributed data with equal variance was tested for significant differences using ANOVA. All other data was tested using ANOVA on RANKs. Post¨Hoc testing was 11 performed all pairwise using the Student-Newman-Keuls Method. All statistical tests were 12 performed using Sigma Stat v3.5.

23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 [00272] Results 2 [00273]
Comparison of Carriers: MicroCT results indicated that CSD carriers produced 3 larger ossicles than either of the calcium phosphate based carriers by total volume (Table 18).
4 There was also a trend for the CSD ossicles to contain more new bone than either of the calcium phosphate containing carriers (Table 19).
6 [00274] Table 18: Total Volume (mm3) '-*`.*Iitaiii?";-"Ntatarkx CSD(B)+P407 Calcium sulphate 209 70 HAp(B)+P407 Hydroxyapatite 135 28 BCP(B)+P407 Biphasic Calcium phosphate 158 22 P(ANOVA on RANKS) 0.017 8 [00275] Post Hoc Test: All pairwise multiple comparison procedures (Student-Newman-Keuls 9 Method). CSD vs HAp (P<0.05); CSD vs BCP (P<0.05); HAp vs BCP (no significant difference).
[00276] Table 19: Adjusted Bone Volume (mm3) !:2:'jP1INnCi:44$';';11X6:ci4T,',-CSD(B)+P407 Calcium sulphate 98.1 41.2 HAp(B)+P407 Hydroxyapatite 67.8 14.1 BCP(B)+P407 Biphasic Calcium phosphate 77.5 17.8 P(ANOVA on RANKS) 0.12 12 [00277] Histology: Histological evaluation indicated that for all bioimplants the ossicles 13 primarily comprised a shell of bone surrounding a mixture of bone, cartilage and marrow tissue.
14 There were no signs of inflammation in any of the implants.
[00278] Residual calcium phosphate granules were visible in the bioimplants containing 16 BCP, or HAp while CSD appears to be rapidly resorbed with only a few CAS
granules seen.
17 [00279] These results show that when BMP-2 was lyophilized onto CSD
carriers the CSD-18 P407 bioimplant produced larger bone ossicles containing more bone than the other carriers 19 onto which BMP-2 was lyophilized.

23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 [00280]
2 [00281] EXAMPLE 16: Production of a bioimplant having increased potency by 3 distributing the BMP both onto the carrier and into the delivery vehicle compared to 4 bioim plants where the BMP was only on the carrier.
[00282] The present example describes the evaluation of calcium sulphate and calcium 6 phosphate individually and as a mixture, as carriers of BMP that might improve bone production 7 relative to ACS in mouse muscle pouch assays, wherein a preferred ratio of BMP on carrier 8 relative to the delivery vehicle (i.e. P407) and a preferred ratio of calcium sulphate to biphasic 9 calcium phosphate is determined.
[00283] Materials and Methods 11 [00284] Experimental Design: To identify a carrier with relatively high bone producing 12 capacity mixtures of calcium sulphate dehydrate (CSD) and biphasic calcium phosphate (BCP) 13 in the ratios of 1:0, 3:1, 1:1 and 0:1 were tested. Further, the ratio of BMP on carrier versus in 14 the F127 delivery vehicle was varied such that ratios of 100:0, 90:10 and 70:30 CSD:BCP were tested. Each variable was tested in a bioimplant wherein the ratio of BMP to implant volume 16 was 40 pg:-50p1, based on a goal of <1mg/cc and wherein the ratio of carrier to F127 was 17 30:45. Experimental design is further set forth in Table 20.
18 [00285] Table 20: Experimental design.
Gp (side Name CSD BCP F127 BMP CARRIER/P407 aib) (mg) (mg) (pi) (pg) BMP ratio la ACS(B) (Infuse) 80 soak lb ACS 0 2a CSD(B)+F 30 45 40 100/0 2b CSD+F 30 45 0 12 3a CSD(B)+F(B) 30 45 40 70/30 4a BCP(B)+F(B) 30 45 40 70/30 4b BCP+F 30 45 0 12 5a 2:1CSD(B)BCP(B)+F(B) 20 10 45 40 70/30 5b 2:1CSD-BCP+F 20 10 45 0 12 6a 2:1CSD(B)BCP(B)+F(B) 20 10 45 40 90/10 7a 1:1CSD(B)BCP(B)+F(8) 15 15 45 40 70/30 12 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 7b 1:1CSD-BCP-FF 45 8a 1:1CSD(B)BCP(B)+F(B) 15 15 45 40 90/10 1 [00286] Preparation of Materials: Sterile macroporous BCP granules (0.5-1mm diameter) 2 comprising 80% fl-tricalcium phosphate, 20% Hydrmapatite were purchased from Citagenix 3 Inc. (Laval, QC). Sterile CSD granules (0.5-1.2 mm) were prepared by grinding Osteoset 4 pellets (Wright Medical Technology Canada Ltd., Mississauga, ON) and sieving between 1.18mm and 0.5mm sieves.
6 [00287] The Infuse kit was purchased from Medtronic of Canada Ltd.
Infuse BMP-2 was 7 prepared by adding water for injection to the lyophilized rhBMP-2 powder in the Infuse kit. The 8 ACS sponge was cut into pieces of approximate 5x5nnm and placed in Eppendorf capsules.
9 [00288] Induce BMP-2 (1mg/m1) was prepared by Induce Biologics Inc.
A 33% poloxamer 407 (P407) gel was prepared by adding 33g of poloxamer 407 (BASF) to cold water. The 11 solution was then sterilized by autoclaving. The poloxamer gel was kept at 2-8 C after 12 sterilization.
13 [00289] BMP was lyophilized onto the carriers as follows: The required amount of carrier 14 was weighed out and placed into a sterile Eppendorf tube. The desired amount of BMP-2 was added to the carrier and was held at room temperature for 30 minutes prior to freezing. Once 16 frozen the Eppendorf tubes were placed in a bench top lyophilizer and lyophilized overnight. All 17 procedures were performed aseptically to maintain sterility. BMP-P407 samples were prepared 18 in bulk in sterile Eppendorf tubes by adding BMP-2 to BMP at the desired concentration. At the 19 time of surgery the appropriate amount of P407 was pipetted out of the tube.
[00290] Surgical Model: As set forth in Example 15.
21 [00291] Analytical methods: Micro CT and histology analyses were as set forth in Example 22 15.
23 [00292] Statistical Analysis: As the ACS alone did not form ossicles that could be measured 24 they were not included in any statistical analyses.
[00293] The microCT parameters were tested for normality and equal variance.
Normally 26 distributed data with equal variance was tested for significant differences using ANOVA. All 27 other data was tested using ANOVA on RANKs. Post ¨ Hoc testing was performed all pairwise 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 using the Student-Newman-Keuls Method. All statistical tests were performed using Sigma Stat 2 v3.5.
3 [00294] Results 4 [00295] Effect of Distributing BMP between the carrier granules and the P407 gel: When BMP was distributed between the P407 gel and the CSD granules it produced larger ossicles 6 than when all of the BMP was lyophilized onto the CSD (total bone volume). (Group3a >
7 Group2a) (Table 22).
8 [00296] When using the 2:1 CSD-BCP granules more bone was formed when 70%
was 9 lyophilized onto the granules and 30% was in the P407 gel then when 90%
was lyophilized and 10% was in the gel (total bone volume). (Group5a > Group7a) (Table 22).
11 [00297] Effect of using CSD rather than BCP granules: In groups with the same distribution 12 of BMP between the granules and P407 we found that using CSD granules produced larger 13 ossicles than BCP (total bone volume) (Gp 3a > Gp 4a) (Table 20). When COS was mixed with 14 BCP groups with more than 50% CSD in the ratio formed the larger ossicles (Group3a (100CSD) > 5a (67% CSD) > Group 7a (50% CSD) = 4a (0% CSD) (total bone volume) (Table 16 22).
17 [00298] Table 21: MicroCT; total volume of bone produced.

Group Name Mean SD
BMP ratio 1 a ACS(B) (Infuse) soak 200.6 94.1 lb ACS
2a CSD(B)+F 100/0 270.7 52.2 2b CSD+F 164.6 57.9 3a CSD(B)+F(B) 70/30 384.6 68.1 4a BCP(B)+F(B) 70/30 299.1 104.3 4b BCP+F 90.6 81.1 5a 2:1CSD(B)BCP(B)+F(B) 70/30 336.5 125.8 5b 2:1CSD-BCP+F 121.2 81.6 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 6a 2:1CSD(B)BCP(B)+F(B) 90/10 259.2 45.1 7a 1:1CSD(B)BCP(B)+F(B) 70/30 275.6 97.1 7b 1:1CSD-BCP+F 137.9 53.5 8a 1:1CSD(B)BCP(B)+F(B) 90/10 269.7 53.9 P value (ANOVA on RANKS) <0.001 2 [00299] Table 22: Post Hoc Test (comparison of BMP containing groups in total volume 3 analysis). All Pairwise Multiple Comparison Procedures (Student-Newman-Keuls Method).

Comparison Diff of Ranks q P<0.05 3a-TV vs 1aTV 811.000 8.404 Yes 3a-TV vs 6a-TV 510.000 6.036 Yes 3a-TV vs 7a-TV 491.000 6.773 Yes 3a-TV vs 8a-TV 451.000 7.455 Yes 3a-TV vs 2aTV 436.000 8.990 Yes 3a-TV vs 4a-TV 396.500 10.864 Yes 3a-TV vs 5a-TV 203.000 8.287 Yes 5a-TV vs 1aTV 608.000 7.195 Yes 5a-TV vs 6a-TV 307.000 4.235 Yes 5a-TV vs 7a-TV 288.000 4.760 Yes 5a-TV vs 8a-TV 248.000 5.114 Yes 5a-TV vs 2aTV 233.000 6.384 Yes 5a-TV vs 4a-TV 193.500 7.900 Yes 2aTV vs 1aTV 375.000 6.199 Yes 4a-TV vs 1aTV 414.500 5.717 Yes 6a-TV vs 1aTV 301.000 12.288 Yes 7a-TV vs 1a-R/ 320.000 8.768 Yes 8a-TV vs 1aTV 360.000 7.423 Yes 6 [00300] Effect of Distributing BMP between the granules and the P407 gel:
When BMP was 7 distributed between the P407 gel and the CSD granules it produced more bone than when all of 8 the BMP was lyophilized onto the CSD (adjusted bone volume). (Gp3a >
Gp2a) (Table 24).
9 [00301] Effect of using CSD rather than BCP granules: In groups with the same distribution of BMP between the granules and P407 we found that using CSD granules produced larger 11 ossicles than BCP (adjusted bone volume) (Gp 3a > Gp 4a) (Table 24).
When CDS was mixed 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 1 with BCP groups with more than 50% CSD in the ratio formed the larger ossicles (adjusted bone 2 volume) (Gp3a (100CSD) > 5a (67% CSD) > 4a (0% CSD) (Table 24).
3 [00302] Table 23: Adjusted Bone Volume (aBV). All pairwise multiple comparison procedures 4 (Student-Newman-Keuls Method).

Group Name Mean SD
BMP ratio 1a ACS(B) (Infuse) soak 75.4 62.6 lb ACS
2a CSD(B)+F 100/0 115.4 34.0 2b CSD+F 68.8 27.4 3a CSD(B)+F(B) 70/30 163.3 39.0 4a BCP(B)+F(B) 70/30 101.3 35.7 4b BCP+F 23.6 13.8 5a 2:1CSD(B)BCP(B)+F(B) 70/30 129.8 45.8 5b 2:1CSD-BCP+F 46.7 29.0 6a 2:1CSD(B)BCP(B)+F(B) 90/10 114.1 41.7 7a 1:1CSD(B)BCP(B)+F(B) 70/30 111.7 26.8 7b 1:1CSD-BCP+F 67.0 23.4 8a 1:1CSD(B)BCP(B)+F(B) 90/10 112.9 34.5 P value (ANOVA on RANKS) <0.001 6 [00303] Table 24: Post Hoc test (comparison of BMP containing groups).
All pairvvise 7 multiple comparison procedures (Student-Newman-Keuls Method) :
Comparison Diff of Ranks q P<0.05 3a- aBV vs 1a - aBV 809.000 8.384 Yes 3a- aBV vs 4a - aBV 569.000 6.734 Yes 3a- aBV vs 8a aBV 421.000 5.807 Yes 3a- aBV vs 7a - aBV 408.000 6.744 Yes 3a- aBV vs 6a - aBV 400.000 8.248 Yes 3a- aBV vs 2a - aBV 386.000 10.576 Yes 3a- aBV vs 5a aBV 235.000 9.594 Yes 23580844.2 CA 2,868,992 Blakes Ref: 75312/00030 5a - aBV vs la-aBV 574.000 6.793 Yes 5a - aBV vs 4a - aBV 334.000 4.607 Yes 2a-aBV vs la-aBV _ 423.000 5.835 Yes 4a - aBV vs la - aBV 240.000 9.798 Yes 6a - aBV vs la - aBV 409.000 6.761 Yes 7a - aBV vs la-aBV 401.000 8.268 Yes 8a - aBV vs la-aBV 388.000 10.631 Yes 2 [00304] Histological evaluation indicated that for all bioimplants the ossicles primarily 3 comprised a shell of bone surrounding a mixture of bone, cartilage and marrow tissue. There 4 were no signs of inflammation in any of the implants.
[00305] Residual calcium phosphate granules were visible in the Induce Bioimplants 6 containing BCP, while calcium sulphate appears to be undergoing rapid resorption with only a 7 few CAS granules seen. Bone was seen forming directly onto and into the CAS and BCP
8 granules (FIG. 10A-B).
9 [00306] Discussion [00307] Results from this study show that when BMP was distributed between being 11 lyophilized onto carrier granules and mixed into the P407 gel, (a distribution which results in a 12 multiphasic release of BMP) larger ossicles with more bone were produced than when all of the 13 BMP was lyophilized onto the granules which were subsequently mixed with P407 gel at the 14 time of surgery [00308] A 70/30 distribution between granules and P407 gel produced larger ossicles with 16 more bone than a 90/10 distribution between granules and P407 gel.
17 [00309] In groups with an equal distribution of BMP between the granules and P407 CSD
18 granules produced larger ossicles with more bone than similarly sized BCP granules, despite 19 CSD-produced ossicles having a larger surface area due to being porous (BCP granules were solid). When CSD can be mixed with BCP bioimplants with 67% or more CSD
granules 21 produced larger ossicles than those with 50% or fewer CSD granules.

23580844.2

Claims (53)

WHAT IS CLAIMED IS:

1. A composition for multiphasic release of growth factors at a treatment site, the composition comprising:
(a) a delivery vehicle comprising a polymer in the form of a liquid or gel and at least one first growth factor, the delivery vehicle being adapted to release the at least one first growth factor in an initial release profile over a first time period; and (b) a carrier comprising a plurality of particles having at least one second growth factor on the surfaces thereof, the carrier being adapted to release the at least one second growth factor in a sustained release profile over a second time period, wherein the second time period is of a longer duration than the first time period, and wherein the carrier comprises calcium sulphate dihydrate particles.

2. The composition according to claim 1, wherein the carrier further comprises calcium phosphate particles.

3. The composition according to claim 2, wherein the weight ratio of calcium sulphate dihydrate to calcium phosphate particles is from about 1:1 to about 2:1.

4. The composition according to any one of claims 1 to 3, wherein the polymer is poloxamer 407.

5. The composition according to any one of claims 1 to 4, wherein the particles of the carrier are combined with the delivery vehicle.

6. The composition according to any one of claims 1 to 5, wherein the first time period comprises hours or days and/or wherein the second time period comprises days or weeks.

7. The composition according to any one of claims 1 to 6, wherein the at least one first growth factor and the at least one second growth factor are the same or different.

8. The composition according to any one of claims 1 to 7, wherein the delivery vehicle cornprises at least 10% of the total amount of the growth factors and the carrier comprises at least 50% of the total amount of the growth factors.

9. The composition according to any one of claims 1 to 8, wherein the delivery vehicle to carrier ratio is from about 0.5:1 to about 4:1 (v/v).

10. A composition comprising a delivery vehicle and a carrier for use in a method of multiphasic release of growth factors, the method comprising:
- delivering at least one first growth factor in an initial release profile over a first time period by means of a delivery vehicle; and - delivering at least one second growth factor in a sustained release profile over a second time period by means of a carrier, wherein the second time period is of a longer duration than the first time period;
wherein the delivery vehicle comprising a polymer in the form of a liquid or gel and the at least one first growth factor, and wherein the carrier comprises a plurality of particles having the at least one second growth factor on the surfaces thereof, and wherein the carrier comprises calcium sulphate dihydrate particles or a mixture of calcium sulphate dihydrate particles and calcium phosphate particles.

11. The composition according to claim 10, wherein the particles of the carrier are combined with the delivery vehicle.

12. The composition according to claim 10 or 11, wherein the polymer is poloxamer 407.

13. The composition according to any one of claims 10 to 12, wherein the first time period comprises hours or days and/or wherein the second time period comprises days or weeks.

14. The composition according to any one of claims 10 to 13, wherein the at least one first growth factor and the at least one second growth factor are the same or different, and wherein the delivery vehicle comprises at least 10% of the total amount of the growth factors and the carrier comprises at least 50% of the total amount of the growth factors.

15. The composition according to any one of claims 1 to 14, wherein the at least one second growth factor is applied and lyophilized onto the carrier particles.

16. The composition according to any one of claims 1 to 15, wherein at least one of the growth factors is a bone morphogenetic protein (BMP).

17. The composition according to claim 16, wherein the bone morphogenetic protein is BMP-2 or BMP-7.

18. The composition according to claim 17, wherein the carrier particles are dispersed within the delivery vehicle.

19. The composition according to any one of claims 1 to 18, wherein the delivery vehicle is adapted to release at least 80% of the at least one first growth factor within a period of 72 hours.

20. A system for multiphasic release of growth factors at a treatment site, the system comprising:
(a) a delivery vehicle comprising at least one first growth factor and a polymer, the delivery vehicle being adapted to release the at least one first growth factor in an initial release profile over a first time period; and (b) a carrier comprising a plurality of particles having at least one second growth factor on the surfaces thereof, the carrier being adapted to release the at least one second growth factor in a sustained release profile over a second time period, and wherein the carrier comprises calcium sulphate dihydrate particles.

21. The system according to claim 20, wherein the carrier further comprises calcium phosphate particles,

22. The system according to claim 21, wherein the weight ratio of calcium sulphate dihydrate to calcium phosphate particles is about 1:1 or 2:1.

23. The system according to any one of claims 20 to 22, wherein the polymer is poloxamer 407.

24. The system according to any one of claims 20 to 23, wherein the particles of the carrier are combined with the delivery vehicle.

25. The system according to any one of claims 20 to 24, wherein the first time period comprises hours or days and/or wherein the second time period comprises days or weeks.

26. The system according to any one of claims 20 to 25, wherein the at least one first growth factor and the at least one second growth factor are the same.

27. The system according to any one of claims 20 to 26, wherein the delivery vehicle comprises at least 10% of the total amount of the growth factors and .the carrier comprises at least 50% of the total amount of the growth factors.

28. The system according to any one of claims 20 to 27, wherein the delivery vehicle to carrier ratio is from about 0.5:1 to about 4:1 (v/v).

29. The system according to any one of claims 20 to 28, wherein the at least one second growth factor is applied to the carrier and then lyophilized onto the carrier.

30. A system comprising a delivery vehicle and a carrier for use in a method of multiphasic release of growth factors, the method comprising:
- delivering at least one first growth factor in an initial release profile over a first time period by means of a delivery vehicle; and - delivering at least one second growth factor in a sustained release profile over a second time period by means of a carrier;
wherein the delivery vehicle comprises the at least one first growth factor and a polymer, and wherein the carrier comprises a plurality of particles having the at least one second growth factor on the surfaces thereof, and wherein the carrier comprises calcium sulphate dihydrate particles or a mixture of calcium sulphate dihydrate particles and calcium phosphate particles.

31. The system for use according to claim 30, wherein the particles of the carrier are combined with the delivery vehicle.

32. The system for use according to claim 30 or 31, wherein the polymer is poloxamer 407.

33. The system for use according to any one of claims 30 to 32, wherein the first time period comprises hours or days and/or wherein the second time period comprises days or weeks.

34. The system for use according to any one of claims 30 to 33, wherein the at least one first growth factor and the at least one second growth factor are the same and wherein the delivery vehicle comprises at least 10% of the total amount of the growth factors and the carrier comprises at least 50% of the total amount of the growth factors.

35. The system of any one of claims 20 to 34, wherein the delivery vehicle is adapted to release at least 80% of the at least one first growth factor within a period of 72 hours.

36. The system according to any one of claims 20 to 35, wherein at least one of the growth factors is a bone morphogenetic protein (BMP).

37. The composition according to claim 36, wherein the bone morphogenetic protein is BMP-2 or BMP-7.

38. A system for multiphasic release of growth factors at a treatment site, the system comprising:
a) a delivery vehicle comprising a polymer comprising poloxamer 407 and at least one first growth factor, wherein the delivery vehicle is adapted to release the at least one first growth factor in an initial release profile over a first time period; and, b) a carrier comprising a plurality of particles having at least one second growth factor on the surfaces thereof, the carrier being adapted to release the at least one second growth factor over a second time period, wherein the second time period is of a longer duration than the first time period, and wherein the carrier comprises calcium sulphate dihydrate particles.

39. The system according to claim 38, wherein the carrier further comprises calcium phosphate particles.

40 The system according to claim 38 or 39, wherein the particles of the carrier are dispersed within the delivery vehicle.

41. The system according to any one of claims 38 to 40, wherein the first time period comprises hours or days.

42. The system according to any one of claims 38 to 41, wherein the second time period comprises days or weeks.

43. The system according to any one of claims 38 to 42, wherein the carrier releases the at least one second growth factor in a sustained release profile over the second time period.

44. The system according to any one of claims 38 to 43, wherein the at least one first growth factor and the at least one second growth factor are the same.

45. The system according to claim 44, wherein the growth factor is bone morphogenetic protein 2 (BMP-2).

46. The system according to claim 44 or 45, wherein the delivery vehicle is adapted to release at least 10% of the total amount of the growth factor during the first time period and the carrier is adapted to release at least 50% of the total amount of the growth factor during the second time period.

47. The system according to any one of claims 38 to 46, wherein the delivery vehicle to carrier ratio is from 0.5:1 to 4:1 (v/v).

48. The system according to any one of claims 38 to 47, wherein the at least one second growth factor is applied as a solution to the carrier and then lyophilized onto the carrier.

49. The system according to claim 48, wherein the concentration of the at least one second growth factor in the solution is from 0.5 mg/ml to 2 mg/ml.

50. The system according to any one of claims 38 to 49, wherein the delivery vehicle is adapted to release at least 80% of the at least one first growth factor within a period of 72 hours.

51. The system according to any one of claims 38 to 50, wherein the delivery vehicle is in the form of a liquid or a gel.

52. The composition according to any one of claims 10 to 14, wherein the delivery vehicle to carrier ratio is from about 0.5:1 to about 4:1 (v/v).

53. The system according to any one of claims 30 to 34, wherein the delivery vehicle to carrier ratio is from about 0.5:1 to about 4:1 (v/v).